Cancer therapy sensitizer

ABSTRACT

The present invention relates to compositions and methods for sensitizing cancer therapy. The invention provides such compositions comprising a SPARC family polypeptide or polynucleotide, as well as recombinant cells containing a SPARC family polypeptide or polynucleotide. The compositions and methods of the invention are useful in in vitro study of cancer therapy resistance, as well as ex vivo and in vivo therapy of cancer.

RELATED APPLICATION(S)

This application is a continuation of Application No. PCT/US04/000901,filed Jan. 14, 2004, which claims the benefit of U.S. ProvisionalApplication No. 60/440,009, filed on Jan. 14, 2003. The entire teachingsof the above applications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to cancer therapy sensitizing compositions andmethods.

BACKGROUND

The reason for cancer treatment failures following induction withchemotherapy or radiation therapy is still unclear. Many factors havebeen implicated in therapeutic resistance, such as upregulation ofefflux pumps from multidrug resistance family (MDR) p-glycoprotein andother non-classical MDR proteins (multidrug resistance-associatedprotein, MRP; lung resistance protein, LRP) have been described in avariety of cancers (Lehnert, M. Anticancer Res 1998; 18:2225-2226;Ringborg, U. and Platz, A. Acta Oncol 1996; 5:76-80; Shea, T. C.,Kelley, S. L., and Henner, W. D. Cancer Res 1998; 48:527-533).Unfortunately, many tumors that are intrinsically resistant tochemotherapy, such as the gastrointestinal malignancies, have relativelylow levels of expression of the MDR genes. For example, only 23% ofprimary colorectal tumors express MRP and 65% express p-glycoprotein(Filipits M, Suchomel R W, Dekan G, Stigilbauer W, Haider K, Depisch D,Pirker R. Br. J Cancer 1997; 75: 208-212). Therefore, resistance totherapeutic agents cannot be explained solely on the basis of activationand up-regulation of known MDR genes. Studies have also shown thatgenetic mutations responsible for tumorigenesis may also contribute todrug resistance. For example, loss of DNA mismatch repair (MMR) genesfound in hereditary non-polyposis colorectal cancer (HNPCC), have beenassociated with a more rapid emergence of clinical drug resistance (delas Alas MM, S Aebi, D Fink, S B Howell, G Los. J Natl Canc Inst 1997;89:1537-41; Lin X, Howell S B (1999). Mol Pharmacol 56:390-5). Mutationsin the K-ras gene, detected in approximately 40% of adenomatous polypsand adenocarcinomas, are associated with an increased relapse rate,mortality and a poor chemotherapeutic response (Arber N, I. Shapira, J.Ratan et al. Gastroenterology 2000; 118:1045-1050). Genes involved incell cycle regulation, such as p21 and p27, have been shown to protecttumors from undergoing apoptosis elicited by various anticancer agents(Waldman T, Lengauer C, Kinzler K W, Vogelstein B. Nature 1996,381:713-716; St. Croix B, Florenes V A, Rak J W, Flanagan M,Bhattacharya N, slingerland J M, Kerbel R S. Nature Med 1996,2:1204-1210). In addition, cell adhesion molecules, such as E-cadherin,confer resistance to cells when exposed to chemotherapeutic agents(Skoudy A, Llosas M D, Garcia de Herreros A. Biochem J 1996).

The mechanisms involved in therapeutic resistance therefore appear to bevery complex. Recent evidence suggests that the selectivity ofchemotherapy for the relatively few tumors ever cured by drugs depends,to a large extent, upon their easy susceptibility to undergo apoptosis,i.e., to kill themselves (Makin G, Expert Opin Ther Targets. 20026(1):73-84; Johnstone R W, Ruefli A A, Lowe S W, Cell. 2002108(2):153-64; Kamesaki H, Int J Hematol. 1998 68(1):29-43).

Secreted protein acidic and rich in cystein (SPARC) belongs to a familyof extracellular proteins, called matricellular proteins. Since itsidentification and cloning, the functional role of SPARC remainsunclear. Its high evolutionary conservation suggests an importantphysiological role for this protein (Iruela-Arispe M L, Lane T F,Redmond D, Reilly M, Bolender R P, Kavanagh T J, Sage E H. Mol BiolCell. 1995 March; 6(3):327-43). Initial studies showed that SPARC isimportant in bone mineralization (Termine J D, Kleinman H K, Whitson SW, Conn K M, McGarvey M L, Martin G R. Cell. 1981 October; 26(1 Pt1):99-105). While SPARC is expressed at high levels in bone tissue, itis also distributed widely in other tissues and cell types (Maillard,C., et al., Bone, 13:257-264 (1992)). Its role has been expanded toinclude tissue remodeling (Latvala T, Puolakkainen P, Vesaluoma M, TervoT. Exp Eye Res. 1996 November; 63(5):579-84; Kelm R J Jr, Swords N A,Orfeo T, Mann K G. J Biol Chem. 1994 Dec. 2; 269(48):30147-53);endothelial cell migration (Hasselaar P, Sage E H. J Cell Biochem. 1992July; 49(3):272-83), morphogenesis (Mason L T, Murphy D, Munke M,Francke U, Elliott R W, Hogan B L. EMBO J. 1986 August; 5(8):1831-7;Strandjord T P, Sage E H, Clark J G. Am J Respir Cell Mol Biol. 1995September; 13(3):279-87), and angiogenesis (Kupprion C, Motamed K, SageE H. J Biol Chem. 1998 Nov. 6; 273(45):29635-40; Lane T F, Iruela-ArispeM L, Johnson R S, Sage E H. J Cell Biol. 1994 May; 125(4):929-43). SPARChas also been shown to have an antiproliferative effect on endothelialcells, mesangial cells, fibroblasts and smooth muscle cells (Sage E H.Biochem Cell Biol. 1992 July; 70(7):579-92).

Experiments in vitro have also identified SPARC in tumors (Schulz, A.,et al., Am. J. Pathol., 132:233-238 (1988); Porter, P. L., et al., J.Histochem. Cytochem., 43:791-800 (1995)). There is conflicting evidencethat SPARC can function either as an oncogene, as suggested by studiesin melanoma (Ledda M F, Adris S, Bravo A T, Kairiyama C, Bover L,Chernajovsky Y, Mordoh J, Podhajcer O L. Nat Med. 1997 February;3(2):171-6) or as a tumor suppressor, as demonstrated by its stronginhibition of growth in vJun-ml and v-Src-transformed chicken embryofibroblasts (Vial E, Castellazzi M. Oncogene. 2000 Mar. 30;19(14):1772-82). Although the growth inhibitory properties of SPARC havebeen mainly shown in primary cells, such as endothelial, fibroblast,mesangial and smooth muscle cells, this may also contribute to the roleof SPARC in tumorigenesis. SPARC has also been shown to have tumorinvasive properties. Variable SPARC expression has been observed in avariety of cancers. Higher levels of expression have been detected inbreast cancer (Bellahcene A, Castronovo V. Am J Pathol. 1995 January;146(1):95-100), esophageal cancer (Porte H, Triboulet J P, Kotelevets L,Carrat F, Prevot S, Nordlinger B, DiGioia Y, Wurtz A, Comoglio P,Gespach C, Chastre E. Clin Cancer Res. 1998 June; 4(6):1375-82),hepatocellular carcinoma (Le Bail B, Faouzi S, Boussarie L, Guirouilh J,Blanc J F, Caries J, Bioulac-Sage P, Balabaud C, Rosenbaum J. J Pathol.1999 September; 189(1):46-52), and prostate (Thomas R, True L D, BassukJ A, Lange P H, Vessella R L. Clin Cancer Res 2000; 6:1140-1149).However, conflicting results have been seen with ovarian cancers (BrownT J, Shaw P A, Karp X, Huynh M H, Begley, Ringuette M J. Gynecol Oncol1999; 75: 25-33; Paley P J, Goff B A, Gown A M, Greer B E, Sage E H.Gynecol Oncol 2000; 78: 336-341; Yiu G K, Chan W Y, Ng S W, Chan P S,Cheung K K, Berkowitz R S, Mok S C. Am J Pathol 2001; 159:609-622), andcolorectal cancers (Porte H, Chastre E, Prevot 5, Nordlinger B, EmpereurS, Basset P, Chambon P, Gespach C. Int J Cancer 1995; 64: 70-75; LussierC, Sodek J, Beaulieu J F. J Cell Biochem. 2001; 81(3):463-76).

Recently, SPARC has been suggested to be involved in inducing apoptosisof ovarian cancer cells (Yiu G K, Chan W Y, Ng S W, Chan P S, Cheung KK, Berkowitz R S, Mok S C. Am J Pathol 2001; 159:609-622). Yiu et al.(2001, supra) has showed that there was downregulation of SPARCexpression following malignant transformation, and that there wereanti-proliferative properties of SPARC on both normal ovarian and cancercells. Yiu et al. (2001, supra) further provided additional evidencethat exogenous exposure to SPARC alone was capable of inducing apoptosisin ovarian cancer cells. However, human pathological specimens of tumorswith high SPARC expression levels have not been shown to have highernumber of apoptotic cells.

WO0202771 discloses a novel hSPARC-h1 polypeptide and its potentialapplications in tissue remodeling, tissue repair and general modulationof various growth factor activities.

U.S. Pat. No. 6,387,664 provides a SPARC fusion protein obtainable byfusing SPARC to thioredoxin which can be used for basic research inneurobiology and/or for treating various neuropathologies.

U.S. Pat. No. 6,239,326 provides a SPARC-deficient transgenic mousemodel for testing drugs in promoting or retarding wound healing andtreating or preventing cataracts, diabetes mellitus or osteoporosis.

All references cited herein above and throughout the specification,including patents, patent applications, are hereby incorporated byreference in their entirety.

SUMMARY OF THE INVENTION

The invention is based on the discovery that SPARC sentitizes cancertherapy.

The present invention provides compositions and methods for sensitizingcancer therapeutic treatment.

The present invention provides a composition comprising a SPARC familypolypeptide and a chemotherapy agent.

The present invention provides a composition comprising a SPARC familypolypeptide and a chemotherapy-resistant cell.

The present invention provides a chemotherapy-resistant cell comprisinga recombinant SPARC family polynucleotide.

The present invention provides a recombinant cell comprising aheterologous transcription control region operatively associated with aSPARC family polynucleotide.

In another aspect, the present invention provides a method for in vivosensitizing a mammal to a therapeutic treatment, the method comprisingadministrating to the mammal diagnosed with cancer an effective amountof a SPARC family polypeptide or a polynucleotide encoding a SPARCpolypeptide.

The present invention provides a method for ex vivo sensitizing a mammaldiagnosed with cancer to a therapeutic treatment, the method comprising:administering to a mammal an effective amount of a cell comprising aSPARC family polypeptide or a polynucleotide encoding a SPARC familypolypeptide; wherein the cell produces an increased amount of the SPARCpolypeptide.

The present invention also provides a method for ex vivo sensitizing amammal diagnosed with cancer to a therapeutic treatment, the methodcomprising: (1) Obtaining a cancer sample from the mammal; (2)contacting the cancer sample with an effective amount of a compositioncomprising a SPARC family polypeptide or a polynucleotide encoding aSPARC polypeptide; and (3) returning the cancer sample after thecontacting of (2) to the mammal.

The present invention further provides a method for sensitizing a cancersample to a therapeutic treatment, the method comprising contacting thecancer sample with an effective amount of a composition comprising aSPARC family polypeptide or a polynucleotide encoding a SPARCpolypeptide.

In one embodiment of the invention, the cancer sample is a cell ortissue sample.

In another embodiment of the invention, the cancer sample is transfectedor infected with the polynucleotide of (e)-(f).

The present invention provides a method for evaluating a first cancercell for its resistance to a therapeutic treatment, comprising: (a)measuring the expression level of a SPARC family mRNA or polypeptide, orthe extracellular level of a SPARC family polypeptide in the firstcancer cell; and (b) comparing the expression level or the extracellularlevel obtained in (a) with the expression level of the SPARC family mRNAor polypeptide, or the extracellular level of the SPARC familypolypeptide in a second cancer cell which does not exhibit resistance tothe therapeutic treatment; wherein a lower level of expression orextracellular level in (a) is indicative of the first cancer cell beingresistant to the therapeutic treatment.

The present invention provides a method for evaluating a first cancercell for its resistance to a therapeutic treatment, comprising: (a)measuring expression level of a SPARC family mRNA or polypeptide, orextracellular level of a SPARC family polypeptide in the first cancersample; (b) measuring expression level of the SPARC family mRNA orpolypeptide, or extracellular level of the SPARC family polypeptide in asecond cancer sample which does not exhibit resistance to thetherapeutic treatment; (c) comparing the expression levels or theextracellular levels obtained in (a) and (b), where a lower level ofexpression or extracellular level in (a) is indicative of the firstcancer sample being resistant to the therapeutic treatment.

In one embodiment, the first sample is from a first mammal and the lowerlevel of expression or extracellular level in (a) is further indicativeof the first mammal being resistant to the therapeutic treatment.

In another embodiment, the second cancer sample is from a first mammalwho provides the first cancer sample.

In yet another embodiment, the second cancer sample is from a secondmammal who is different from the first mammal providing the first cancersample.

Preferably, the first mammal and the second mammal are diagnosed withthe same cancer.

In one embodiment, the expression level of the SPARC family mRNA ismeasured by polymerase chain reaction, DNA microarray or northern blot.

In one embodiment, the expression or extracellular level of the SPARCfamily polypeptide is measured by Immuno Blotting or Enzyme-LinkedImmunosorbent Assay (Elisa).

The present invention provides a method for identifying an agent whichmodulates a SPARC family mRNA or polypeptide expression, or a SPARCfamily polypeptide secretion, comprising: (a) measuring expression levelof the SPARC family mRNA or polypeptide, or extracellular level of theSPARC family polypeptide in a sample; (b) contacting a candidate agentwith the sample; (c) after the contacting of (b), measuring expressionor extracellular level of the SPARC family mRNA or polypeptide, orextracellular level of the SPARC family polypeptide in the sample of(b); (d) comparing the expression levels or the extracellular levels in(a) and (c), where a differential level of expression or extracellularlevel in (a) and (c) indicates the candidate agent being an agent whichmodulates the SPARC family mRNA or polypeptide expression, or the SPARCfamily polypeptide secretion.

The present invention also provides a method for identifying an agentwhich sensitizes a cancer sample to a therapeutic treatment, comprising:(a) measuring expression level of a SPARC family mRNA or polypeptide, orextracellular level of a SPARC family polypeptide in the cancer sample;(b) contacting a candidate agent with the cancer sample; (c) after thecontacting of (b), measuring expression level of the SPARC family mRNAor polypeptide, or extracellular level of the SPARC family polypeptidein the cancer sample of (b); (d) comparing the expression levels or theextracellular levels obtained in (a) and (c), where an increased levelof expression or extracellular level in (c) indicates the candidateagent being an agent which sensitizes a cancer sample to a therapeutictreatment.

In one embodiment, the cancer sample of the subject method is from amammal diagnosed with cancer, and the increased level of expression orextracellular level is further indicative of the candidate agent beingan agent which sensitizes the mammal to a therapeutic treatment.

The present invention provides a method for determining a therapeutictreatment protocol for a first mammal diagnosed with cancer, comprising:(a) determining if expression of a SPARC family mRNA or polypeptide orextracellular level of a SPARC family polypeptide is lower in a firstsample from the first mammal than a second sample which does not exhibitresistance to the therapeutic treatment; and (b) if (a) is positive,increasing the strength of the therapeutic treatment to the first mammalso as to increase the response to the treatment.

Preferably, the polynucleotide of (e) or (f) of the subject compositionand method is an expression vector.

More preferably, the expression vector is a plasmid or a viral vector.

In one embodiment, the plasmid vector is pcDNA3.1.

The SPARC family polypeptide or a polynucleotide encoding a SPARC familypolypeptide of the present invention include: (a) a SPARC polypeptidewhich is selected from the group consisting of SEQ ID Nos. 1-17; (b) apolypeptide having an amino acid sequence of at least 60% homology tothe SPARC family polypeptide in (a); (c) a polypeptide fragment of(a)-(b) where the fragment is at least 50 amino acids in length; (d) afusion polypeptide comprising the polypeptide of (a), (b), or (c); (e) apolynucleotide encoding the polypeptide of (a), (b), (c) or the fusionpolypeptide of (d); or (f) a polynucleotide hybridizing to thepolynucleotide of (e) under a stringent hybridization condition.

Preferably, the SPARC family polypeptide or polynucleotide of thepresent invention is selected from: SMOC-1, SPARC, hevin, SC1, QR-1,follistatin-like proteins (TSC-36), testican.

Preferably, the therapeutic agent of the subject composition and methodis a chemotherapy agent or a radiation therapy agent.

More preferably, the therapeutic agent is selected from the groupconsisting of the agents listed in Table 1.

In one embodiment, the mammal of the subject method exhibits resistanceto the therapeutic treatment.

In one embodiment, the therapeutic treatment is chemotherapy orradiation therapy.

In one embodiment, the cancer sample of the subject method is a cell ora tissue sample.

Preferably, the cell or tissue sample is lysed before the measuring ofthe expression or extracellular level of a SPARC family polypeptide orpolynucleotide (e.g., mRNA).

More preferably, a polynucleotide or polypeptide extract is obtainedfrom the cell or tissue sample before the measuring of the expression orextracellular level of a SPARC family polypeptide or polynucleotide(e.g., mRNA).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the invention will be more readily understoodby reference to the following detailed description, taken with referenceto the accompanying drawings:

FIG. 1 is a modular structure of human SPARC and the location andfunctions of synthetic peptides. Three domains and their residue numbersare shown as described in Yan and Sage, 1999, J. Histochem. & Cytochem.47(12):1495-1505.

FIG. 2 is a domain organization of various SPARC family proteins. FSrepresents the follistatin-like domain, TY the thyroglobulin-likedomain, EC the extracellular calcium-binding domain as described inVannahme et al., 2002, J. Biol. Chem. 277(41):37977-37986. Domains withno homology to other proteins are shown as open boxes. Signal peptidesare not shown.

FIG. 3 is a picture showing colony formation assays of chemotherapysensitive and resistant cells according to one embodiment of theinvention.

FIG. 4 is a picture showing TUNEL assays of chemotherapy sensitive andresistant cells according to one embodiment of the invention.

FIGS. 5 (A and B) is a picture showing the decreased level of SPARCpolypeptide in chemotherapy resistant cell lines according to oneembodiment of the invention.

FIG. 6 is a Tunel assay showing the response of the resistant MIP101cells to exogenous SPARC in reversing the resistant phenotype accordingto one embodiment of the invention.

FIG. 7 is a immuno blot assay showing recombinant cells expressing SPARCpolypeptide according to one embodiment of the invention.

FIG. 8 is a FACS analysis showing cell populations induced to undergoapoptosis following exposure to chemotherapeutic agents according to oneembodiment of the invention.

FIG. 9 is a graph showing the percentage of apoptosis of cells followingexposure to chemotherapeutic agents according to one embodiment of theinvention.

FIG. 10 is a graph showing the response of SPARC transfectants tochemotherapy agents according to one embodiment of the invention.

FIG. 11 is a graph presentation showing complete tumor regression inanimals transplanted with SPARC-transfectants following 6 cycles ofchemotherapy in two animals according to one embodiment of theinvention.

FIG. 12 is a figure containing the polynucleotide and polypeptidesequences of the SPARC family members according to one embodiment of theinvention.

FIG. 13 is a figure showing the sequence alignment among different SPARCfamily polypeptides and polynucleotides according to one embodiment ofthe invention.

FIG. 14 shows effect of chemotherapy on tumor xenografts ofSPARC-overexpressing cells according to one embodiment of the invention.

FIG. 15 shows effect of radiation therapy on tumor xenografts ofSPARC-overexpressing cells according to one embodiment of the invention.

FIG. 16 shows treatment of MIP101 tumor xenografts with combinationtherapy with SPARC(s) intraperitoneally according to one embodiment ofthe invention.

FIG. 17 shows treatment of MIP101 tumor xenografts with combinationtherapy with SPARC(s) subcutaneously according to one embodiment of theinvention.

FIG. 18 shows treatment of MIP/5FU tumor xenografts with combinationtherapy with SPARC(s) according to one embodiment of the invention.

FIG. 19 shows human SPARC mRNA and protein levels in colorectal cancercell lines sensitive and resistant to chemotherapy according to oneembodiment of the invention.

FIG. 20 shows SPARC protein expression in human colonic epitheliumaccording to one embodiment of the invention.

FIG. 21 shows assessment of the effect of SPARC in influencing thesensitivity of cells to chemotherapy according to one embodiment of theinvention.

DETAILED DESCRIPTION

The invention is based on the SPARC family and sensitization to cancertherapy.

DEFINITIONS

As used herein, a “SPARC family polypeptide” refers to a polypeptide(including a fragment or variant thereof) of a family of extracellularproteins. This family of extracellular proteins include SPARC and othermembers of the family, such as SMOC-1, hevin, SC1, QR-1,follistatin-like proteins (TSC-36) and testican (see for example,Vannahme et al., (2002), J. Biol. Chem. 277(41): 37977-37986; Johnston,I. G., Paladino, T., Gurd, J. W., and Brown, I. R. (1990) Neuron 2,165-176; Guermah, M., Crisanti, P., Laugier, D., Dezelee, P., Bidou, L.,Pessac, B., and Calothy, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,4503-4507; Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T., and Nose,K. (1993) Eur. J. Biochem. 217, 13-19; Alliel, P. M., Perin, J. P.,Jolles, P., and Bonnet, F. J. (1993) Eur. J. Biochem. 214, 347-350,hereby incorporated by reference.) A SPARC family polypeptide istypically composed of three independently folded domains (Yan and Sage,1999, J. Histochem. & Cytochem., 47(12):1495-1505, hereby incorporatedby reference). The N-terminal domain (e.g., two adjacent N-terminal Glu₃and Glu₄ in SPARC) is negatively charged, the second domain (e.g.,residues 53-137 in SPARC) is homologous to follistatin (FS)1 with 10cysteines in a typical pattern, the C-terminal extracellularcalcium-binding (EC) domain (e.g., residues 138-286 in SPARC) has twoEF-hand calcium-binding motifs, each with a bound calcium ion in thex-ray structure (Maurer, P., Hohenadl, C., Hohenester, E., Göhring, W.,Timpl, R., and Engel, J. (1995) J. Mol. Biol. 253, 347-357; Hohenester,E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., and Engel, J.(1996) Nat. Struct. Biol. 3, 67-73).

The term “SPARC family polypeptide”, according to the present invention,includes the full length polypeptide or a fragment thereof, a wild-typepolypeptide or any variant thereof. A “SPARC family polynucleotide” is apolynucleotide (e.g., DNA or mRNA) molecule encoding a SPARCpolypeptide, or a fragment thereof. (a) a SPARC family polypeptide orgene selected from the group consisting of SEQ ID Nos. 1-17; (b) apolypeptide having an amino acid sequence of at least 60% homology tothe SPARC family polypeptide in (a) or a gene encoding the polypeptideof at least 60% homology; (c) a polypeptide fragment of (a)-(b) whereinthe fragment is at least 50 amino acids in length; (d) a fusionpolypeptide comprising the polypeptide of (a), (b), or (c); (e) apolynucleotide encoding the polypeptide of (a), (b), (c) or the fusionpolypeptide of (d); or (f) a polynucleotide hybridizing to thepolynucleotide of (e) under a stringent hybridization condition.

The term “SPARC” polypeptide refers to SEQ ID Nos. 1-17, and the term“SPARC gene” to SEQ ID Nos. 18-34, the corresponding nucleotidesequences of SEQ ID Nos. 1-17. It is contemplated that variations ofthese sequences which retain the biological activity of SPARC areequivalents of these sequences. The biological activity of the SPARCgene is that it is downregulated in chemotherapy resistant cells. Thegene also may be overexpressed in cells that are sensitive tochemotherapy. The biological activity of the SPARC polypeptide is thatit sensitizes chemotherapy resistant cells to chemotherapy.

With respect to a SPARC family polypeptide member, it is contemplatedthat variations of their sequences which retain the biological activityof the family member are equivalents of these sequences. The biologicalactivity of a SPARC gene family member is that the gene is downregulatedin chemotherapy resistant cells, i.e., expression decreased by at least25%, for example, 30%, 40%, 50%, 75%, 100% (1-fold), 2-fold, 4-fold, or5-fold or more, compared to chemotherapy sensitive cells. The gene alsomay be overexpressed in cells that are sensitive to chemotherapy. Thebiological activity of the SPARC family polypeptide member is that itsensitizes chemotherapy resistant cells to chemotherapy, i.e., increasethe response to chemotherapy by at least 25%, for example, 30%, 40%,50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,15-fold, 20-fold or more, compared to treatment sensitivity in theabsence of the SPARC family polypeptide.

As defined herein, a “tissue” is an aggregate of cells that perform aparticular function in an organism. The term “tissue” as used hereinrefers to cellular material from a particular physiological region. Thecells in a particular tissue may comprise several different cell types.A non-limiting example of this would be brain cells that furthercomprise neurons and glial cells, as well as capillary endothelial cellsand blood cells.

The term “cell type” or “tissue type” refers to the tissue of origin,for example, from which a tumor develops. Such tissues (cells types)include, for example, without limitation, blood, colorectal, breast,esophageal, hepatocellular, prostate, ovarian, thyroid, pancreas,uterus, testis, pituitary, kidney, stomach, esophagus and rectum.

As used herein, the term “cancer” refers to a proliferative disorderdisease caused or characterized by the proliferation of cells which havelost susceptibility to normal growth control. The term “cancer,” as usedin the present application, includes tumors and any other proliferativedisorders. Cancers of the same tissue type originate in the same tissue,and may be divided into different subtypes based on their biologicalcharacteristics. The cancer may be selected from one or more from thegroup consisting of: melanoma, leukemia, astocytoma, glioblastoma,lymphoma, glioma, Hodgkins lymphoma, chronic lymphocyte leukemia andcancer of the pancreas, breast, thyroid, ovary, uterus, testis,pituitary, kidney, stomach, esophagus and rectum.

As used herein, the term “sensitizing” refers to an increasedsensitivity or reduce the resistance of a cancer sample or a mammalresponding to a therapeutic treatment. An increased sensitivity or areduced sensitivity to a therapeutic treatment is measured according toa known method in the art for the particular treatment and methodsdescribed herein below, including, but not limited to, cellproliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, CancerRes 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker RH, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94:161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69:615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R,Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia andLymphoma. Langhorne, P A: Harwood Academic Publishers, 1993: 415-432;Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivityor resistance may also be measured in animal by measuring the tumor sizereduction over a period of time, for example, 6 month for human and 4-6weeks for mouse. A composition or a method sensitizes response to atherapeutic treatment if the increase in treatment sensitivity or thereduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%,70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold,20-fold or more, compared to treatment sensitivity or resistance in theabsence of such composition or method. The determination of sensitivityor resistance to a therapeutic treatment is routine in the art andwithin the skill of an ordinarily skilled clinician.

As used herein, the term “administer” or “administering” refers tointroduce by any means a composition (e.g., a therapeutic agent) intothe body of a mammal in order to prevent or treat a disease or condition(e.g., cancer).

As used herein, the terms “treating”, “treatment”, “therapy” and“therapeutic treatment” as used herein refer to curative therapy,prophylactic therapy, or preventative therapy. An example of“preventative therapy” is the prevention or lessening of a targeteddisease (e.g., cancer) or related condition thereto. Those in need oftreatment include those already with the disease or condition as well asthose prone to have the disease or condition to be prevented. The terms“treating”, “treatment”, “therapy” and “therapeutic treatment” as usedherein also describe the management and care of a mammal for the purposeof combating a disease, or related condition, and includes theadministration of a composition to alleviate the symptoms, side effects,or other complications of the disease, condition. Therapeutic treatmentfor cancer includes, but is not limited to, surgery, chemotherapy,radiation therapy, gene therapy, and immunotherapy.

By “therapeutically effective amount” is meant an amount that relieves(to some extent, as judged by a skilled medical practitioner) one ormore symptoms of the disease or condition in a mammal. Additionally, by“therapeutically effective amount” is meant an amount that returns tonormal, either partially or completely, physiological or biochemicalparameters associated with or causative of a disease or condition. Aclinician skilled in the art can determine the therapeutically effectiveamount of a composition in order to treat or prevent a particulardisease condition, or disorder when it is administered, such asintravenously, subcutaneously, intraperitoneally, orally, or throughinhalation. The precise amount of the composition required to betherapeutically effective will depend upon numerous factors, e.g., suchas the specific activity of the active agent, the delivery deviceemployed, physical characteristics of the agent, purpose for theadministration, in addition to many patient specific considerations. Thedetermination of amount of a composition that must be administered to betherapeutically effective is routine in the art and within the skill ofan ordinarily skilled clinician.

As used herein, the term “agent” or “drug” or “therapeutic agent” refersto a chemical compound, a mixture of chemical compounds, a biologicalmacromolecule, or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian) cells ortissues that are suspected of having therapeutic properties. The agentor drug may be purified, substantially purified or partially purified.An “agent”, according to the present invention, also includes aradiation therapy agent.

As used herein, “modulation” or “modulating” means that adesired/selected response is more efficient (e.g., at least 10%, 20%,40%, 60% or more), more rapid (e.g., at least 10%, 20%, 40%, 60% ormore), greater in magnitude (e.g., at least 10%, 20%, 40%, 60% orgreater), and/or more easily induced (e.g., at least 10%, 20%, 40%, 60%or more) in the presence of an agent than in the absence of the agent.

As used herein, the term “resistance to a therapeutic treatment” refersto an acquired or natural resistance of a cancer sample or a mammal to atherapy, i.e., being nonresponsive to or having reduced or limitedresponse to the therapeutic treatment, e.g., having a reduced responseto a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%,60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,15-fold, 20-fold or more. The reduction in response is measured bycomparing with the same cancer sample or mammal before the resistance isacquired, or by comparing with a different cancer sample or a mammal whois known to have no resistance to the therapeutic treatment. A typicalacquired resistance to chemotherapy is called “multidrug resistance.”The multidrug resistance can be mediated by P-glycoprotein or can bemediated by other mechanisms, or it can occur when a mammal is infectedwith a multi drug-resistant microorganism or a combination ofmicroorganisms. The determination of resistance to a therapeutictreatment is routine in the art and within the skill of an ordinarilyskilled clinician, for example, can be measured by cell proliferativeassays and cell death assays as described herein above under“sensitizing”.

As used herein, the term “chemotherapy” refers to the use of drugs todestroy cancer cells (including leukaemias and lymphomas). There areover 50 different chemotherapy drugs and some are given on their own,but often several drugs may be combined (known as combinationchemotherapy). Chemotherapy may be used alone to treat some types ofcancer. Sometimes it can be used together with other types of treatmentsuch as surgery, radiotherapy, immunotherapy, or a combination thereof.

As used herein, “radiotherapy”, also called “radiation therapy”, refersto the treatment of cancer and other diseases with ionizing radiation.Ionizing radiation deposits energy that injures or destroys cells in thearea being treated (the “target tissue”) by damaging their geneticmaterial, making it impossible for these cells to continue to grow.Although radiation damages both cancer cells and normal cells, thelatter are able to repair themselves and function properly. Radiotherapymay be used to treat localized solid tumors, such as cancers of theskin, tongue, larynx, brain, breast, or uterine cervix. It can also beused to treat leukemia and lymphoma (cancers of the blood-forming cellsand lymphatic system, respectively)

As used herein, the term “treatment protocol” refers to the process ofinforming the decision making for the treatment of a disease. As usedherein, treatment protocol is based on the comparative levels of one ormore cell growth-related polypeptides in a patient's tissue samplerelative to the levels of the same polypeptide(s) in a plurality ofnormal and diseased tissue samples from mammals for whom patientinformation, including treatment approaches and outcomes is available.

As used herein, the term “biological characteristics” refers to thephenotype and/or genotype of one or more cells or tissues, which caninclude cell type, and/or tissue type from which the cell was obtained,morphological features of the cell(s)/tissue(s), and the expression ofbiological molecules within the cell(s)/tissue(s).

As used herein, the term “sample” refers to material derived from thebody of a mammal, including, but not limited to, blood, serum, plasma,urine, nipple aspirate, cerebrospinal fluid, liver, kidney, breast,bone, bone marrow, testes or ovaries and brain, colon, and lung. A“sample,” according to the present invention, may also be cultured cellsand tissues.

As used herein, a “cancer sample” refers to a sample which originatesfrom a cancer, i.e., from a new growth of different or abnormal tissue.A “cancer sample” may be a cell or tissue sample. The cancer cells mayexist as part of the cancer tissue, or may exist as free-floating cellsdetached from the cancer tissue from which they originate. A cancersample, according to the present invention, may be used for in vitro orex vivo testing of cancers.

As used herein, the term “non-cancer sample” refers to cell or tissuesample obtained from a normal tissue. A sample may be judged a non-tumorsample by one of skill in the art on the basis of morphology and otherdiagnostic tests.

As used herein, the term “mammal” refers to a human or other animal,such as farm animals or laboratory animals (e.g. guinea pig or mice).

As used herein, “specific hybridization” or “selective hybridization” or“hybridization under a stringent condition” refers to hybridizationwhich occurs when two polynucleotide sequences are substantiallycomplementary, i.e., there is at least about 60% and preferably, atleast about 80% or 90% identity between the sequences, wherein theregion of identity comprises at least 10 nucleotides. In one embodiment,the sequences hybridize under stringent conditions following incubationof the sequences overnight at 42° C., followed by stringent washes(0.2×SSC at 65° C.). Typically, stringent conditions will be those inwhich the salt concentration is at least about 0.01 to 1.0 M Na ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for short probes (e.g., about 6 to 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH,as calculated using methods routine in the art.

As used herein, the term “homology” refers to the optimal alignment ofsequences (either nucleotides or amino acids), which may be conducted bycomputerized implementations of algorithms. “Homology”, with regard topolynucleotides, for example, may be determined by analysis with BLASTNversion 2.0 using the default parameters. “Homology”, with respect topolypeptides (i.e., amino acids), may be determined using a program,such as BLASTP version 2.2.2 with the default parameters, which alignsthe polypeptides or fragments being compared and determines the extentof amino acid identity or similarity between them. It will beappreciated that amino acid “homology” includes conservativesubstitutions, i.e. those that substitute a given amino acid in apolypeptide by another amino acid of similar characteristics. Typicallyseen as conservative substitutions are the following replacements:replacements of an aliphatic amino acid such as Ala, Val, Leu and Ilewith another aliphatic amino acid; replacement of a Ser with a Thr orvice versa; replacement of an acidic residue such as Asp or Glu withanother acidic residue; replacement of a residue bearing an amide group,such as Asn or Gln, with another residue bearing an amide group;exchange of a basic residue such as Lys or Arg with another basicresidue; and replacement of an aromatic residue such as Phe or Tyr withanother aromatic residue. A “homology of 60% or higher” includes ahomology of, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and up to100% (identical) between two or more nucleotide or amino acid sequences.

As used herein, the term “polynucleotide” includes RNA, cDNA, genomicDNA, synthetic forms, and mixed polymers, both sense and antisensestrands, and may be chemically or biochemically modified or may containnon-natural or derivatized nucleotide bases, as will be readilyappreciated by those skilled in the art. Such modifications include, forexample, labels, methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), and modified linkages (e.g., alpha anomericpolynucleotides, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions.

As used herein, the term “mutation” refers to a change in nucleotidesequence within a gene, or outside the gene in a regulatory sequencecompared to wild type. The change may be a deletion, substitution, pointmutation, mutation of multiple nucleotides, transposition, inversion,frame shift, nonsense mutation or other forms of aberration thatdifferentiate the polynucleotide or protein sequence from that of anormally expressed gene in a functional cell where expression andfunctionality are within the normally occurring range.

“Polypeptide” and “protein” are used interchangeably herein to refer toa polymer of amino acid residues. The term “recombinant protein” refersto a protein that is produced by expression of a recombinant DNAmolecule that encodes the amino acid sequence of the protein.Polynucleotides and recombinantly produced polypeptide, and fragments oranalogs thereof, may be prepared according to methods known in the artand described in Maniatis et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., (1989), Cold Spring Harbor, N.Y., and Berger andKimmel, Methods in Enzymology, Volume 152, Guide to Molecular CloningTechniques (1987), Academic Press, Inc., San Diego, Calif., which areincorporated herein by reference.

As used herein, the term “fragment” when in reference to a polypeptide(as in “a fragment of a given protein”) refers to a shorter portion ofthe polypeptide. The fragment may range in size from four amino acidresidues to the entire amino acid sequence minus one amino acid. In oneembodiment, the present invention contemplates “functional fragments” ofa polypeptide. Such fragments, according to the present invention, are“functional” in that they retain the ability to sensitize a cancersample or cancer mammal to a therapeutic treatment, albeit with perhapslower sensitizing activity than that observed for the full-lengthpolypeptide. Such “fragment” of a polypeptide is preferably greater than10 amino acids in length, and more preferably greater than 50 aminoacids in length, and even more preferably greater than 100 amino acidsin length. A “fragment” of a SPARC family polypeptide, according to thepresent invention, may contain one or more of the three conserveddomains of the SPARC family members, i.e., the Acidic N-terminal domain,the follistatin-like domain, and the extracellular calcium-binding ECdomain.

As used herein, a “variant” of a specific polypeptide refers to apolypeptide substantially similar to either the entire specific peptideor a fragment thereof. By “substantially similar”, it means that thevariant is made to arrive at a final construct which possesses thedesired function, i.e., sensitizing a cancer sample or a mammal to atherapeutic treatment, albeit with perhaps lower sensitizing activity ofthe variant than that observed for the wild-type polypeptide. Suchvariants include, for example, deletions, insertions, or substitutionsof residues within the amino-acid sequence of the specific polypeptide.In addition, a “variant” may also be a fusion polypeptide between aSPARC family polypeptide and a second polypeptide. Any combination ofdeletion, insertion, substitution, and fusion may also be made.

As used herein, “isolated” or “purified” when used in reference to apolynucleotide or a polypeptide means that a naturally occurringnucleotide or amino acid sequence has been removed from its normalcellular environment or is synthesized in a non-natural environment(e.g., artificially synthesized). Thus, an “isolated” or “purified”sequence may be in a cell-free solution or placed in a differentcellular environment. The term “purified” does not imply that thenucleotide or amino acid sequence is the only polynucleotide orpolypeptide present, but that it is essentially free (about 90-95%, upto 99-100% pure) of non-polynucleotide or polypeptide material naturallyassociated with it.

As used herein the term “encoding” refers to the inherent property ofspecific sequences of nucleotides in a polynucleotide, such as a gene ina chromosome or an mRNA, to serve as templates for synthesis of otherpolymers and macromolecules in biological processes having a definedsequence of nucleotides (i.e., rRNA, tRNA, other RNA molecules) or aminoacids and the biological properties resulting therefrom. Thus a geneencodes a protein, if transcription and translation of mRNA produced bythat gene produces the protein in a cell or other biological system.Both the coding strand, the nucleotide sequence of which is identical tothe mRNA sequence and is usually provided in sequence listings, andnon-coding strand, used as the template for transcription, of a gene orcDNA can be referred to as encoding the protein or other product of thatgene or cDNA. A polynucleotide that encodes a protein includes anypolynucleotides that have different nucleotide sequences but encode thesame amino acid sequence of the protein due to the degeneracy of thegenetic code. Polynucleotides and nucleotide sequences that encodeproteins may include introns and may be genomic DNA.

As used herein, the term “vector” refers to a polynucleotide compoundused for introducing exogenous or endogenous polynucleotide into hostcells. A vector comprises a nucleotide sequence which may encode one ormore polypeptide molecules. Plasmids, cosmids, viruses andbacteriophages, in a natural state or which have undergone recombinantengineering, are non-limiting examples of commonly used vectors toprovide recombinant vectors comprising at least one desired isolatedpolynucleotide molecule.

As used herein, the term “transformation” or the term “transfection”refers to a variety of art-recognized techniques for introducingexogenous polynucleotide (e.g., DNA) into a cell. A cell is“transformed” or “transfected” when exogenous DNA has been introducedinside the cell membrane. The terms “transformation” and “transfection”and terms derived from each are used interchangeably.

As used herein, an “expression vector” refers to a recombinantexpression cassette which has a polynucleotide which encodes apolypeptide (i.e., a protein) that can be transcribed and translated bya cell. The expression vector can be a plasmid, virus, or polynucleotidefragment.

The term “expression” refers to the production of a protein ornucleotide sequence in a cell or in a cell-free system, and includestranscription into an RNA product, post-transcriptional modificationand/or translation into a protein product or polypeptide from a DNAencoding that product, as well as possible post-translationalmodifications.

As used herein, the term “comparing the expression level” refers tocomparing the deferential expression of a polynucleotide or apolypeptide in two or more samples.

As used herein, the term “differential expression” refers to bothquantitative, as well as qualitative, differences in a polynucleotide ora polypeptide expression patterns among two or more samples. Apolynucleotide or a polypeptide is said to be “differentially expressed”if its expression is detectable in one sample, but not in anothersample, by known methods for polynucleotide or polypeptide detection(e.g., electrophoresis). A polynucleotide or a polypeptide is also saidto be “differentially expressed” if the quantitative difference of itsexpression (i.e., increase or decrease, measured in μg, μmol or copynumber) between two samples is about 20%, about 30%, about 50%, about70%, about 90% to about 100% (about two-fold) or more, up to andincluding about 1.2 fold, 2.5 fold, 5-fold, 10-fold, 20-fold, 50-fold ormore. A “differentially expressed” gene transcript means a mRNAtranscript that is found in different numbers of copies between two ormore samples.

As used herein, an “increased amount of a SPARC family polypeptide orpolynucleotide” refers to a greater level of expression of at least 20%,e.g., 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or 2-fold, 3-fold, 4-fold,5-fold or more in a cell, compared to a control cell. A cell expressinga SPARC family polypeptide or polynucleotide is said to have an increaseamount of such polypeptide or polynucleotide if the expression is asdefined herein above when compared with a chemotherapy resistant cell.

The term “secreted protein” refers to a protein having at least aportion which is extracellular and includes proteins which arecompletely extracellular (i.e., not attached to a cell). “Level ofsecretion” refers to the level (i.e., amount) of a secreted protein inthe extracellular compartment.

As used herein, the term “proliferation” refers to the rate of celldivision and the ability of a cell to continue to divide. One completecell division process is referred to as a “cycle”. By an “increase incell proliferation” is meant to increase the cell division rate so thatthe cell has a higher rate of cell division compared to normal cells ofthat cell type, or to allow the cell division to continue for morecycles without changing the rate of each cell division, e.g., increaseby 10% or higher (e.g., 20%, 30%, 40% 50%, up to 2 fold, 5 fold, 10 foldor higher. By an “decrease in cell proliferation” is meant to decreasethe cell division rate so that the cell has a lower rate of celldivision compared to normal cells of that cell type, or to reduce thenumber of cycles of the cell division without changing the rate of eachcell division, e.g., decrease by 10% or higher (e.g., 20%, 30%, 40% 50%,up to 2 fold, 5 fold, 10 fold or higher).

The present invention provides compositions and methods for sensitizingcancer therapeutic treatments. Such sensitizing compositions and methodsare particularly useful in enhancing the response of patients who areresistant to a treatment. They are also useful in reducing theside-effects of cancer therapy, for example, by enhancing the responseof a patient to a smaller strength (i.e., dosage) of the treatment. Thecomposition of the present invention may reduce the dosage of atherapeutic treatment agent by at least 20%, for example, 30%, 40%, 50%,and up to 60%.

The SPARC Family Polypeptides and Polynucleotides

In one embodiment, the present invention provides (a) a SPARC familypolypeptide selected from the group consisting of SEQ ID Nos. 1-17; (b)a polypeptide having an amino acid sequence of at least 60% homology tosaid SPARC family polypeptide in (a); (c) a polypeptide fragment of(a)-(b) wherein said fragment is at least 50 amino acids in length, and(d) a fusion polypeptide comprising the polypeptide of (a)-(c).

A SPARC family polypeptide provided by the present invention may be awild-type polypeptide or a variant thereof, it may be the full lengthpolypeptide or a fragment thereof. A SPARC family polypeptide is withinthe scope of the present invention so long as it retains the function ofsensitizing a cancer sample or a patient to a therapeutic treatment,albeit that a lower activity may exist for a variant or a fragmentpolypeptide when compared to the wile-type or full length polypeptide.

Based on sequence homology, several members of the SPARC family havebeen identified, such as SMOC-1 (Vannahme et al., 2002, J. Biol. Chem.277(41):37977-37986), hevin (Bendik I, Schraml P, Ludwig C U, CancerRes. 1998; 58(4):626-9), SC1 (Johnston I G, Paladino T, Gurd J W, BrownI R, Neuron. 1990 4(1):165-76), QR-1, follistatin-like proteins (TSC-36)(Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T. and Nose, K, 1993,Eur. J. Biochem. 217 (1) 13-19) and testican (Alliel, P. M., Perin, J.P., Jolles, P. and Bonnet, F. J, 1993, Eur. J. Biochem. 214 (1),347-350). A SPARC family polypeptide of the present invention includes,but is not limited to, SPARC and any of its identified family membersknown in the art or as described herein.

The alignment of the various polynucleotide sequences permit one skilledin the art to select conserved portions of the proteins (i.e. thoseportions in common between two or more sequences) as well as unconservedportions (i.e. those portions unique to two or more sequences). In oneembodiment, the present invention contemplates conserved fragments 10amino acids in length or greater, and more typically greater than 50amino acids in length. Preferably, the SPARC family polypeptide of thepresent invention contains one or two or three domains (i.e. the AcidicN-terminal domain, the follistatin-like domain, and/or the EC domain),conserved among the SPARC family members.

The therapy-sensitizing fragment or a variant of a wild-type SPARCfamily protein may be delineated by routine sequence manipulations knownto those skilled in the art, including, but not limited to, deletionmutations, point mutations, fusions as described herein below and asdescribed in J. Sambrook, E. F. Fritsch, and T. Maniatis, MolecularCloning: A. Laboratory Manual, 2 Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., CurrentProtocols in Molecular Biology, 1994, incorporated by reference herein.

A mutation in the DNA encoding the variant polypeptide must not alterthe reading frame and preferably will not create complementary regionsthat could produce secondary mRNA structures. At the genetic level thesevariants are prepared by site directed mutagenesis of nucleotides in theDNA encoding the peptide molecule thereby producing DNA encoding thevariant, and thereafter expressing the DNA in recombinant cell culture.

In Vitro Production and Purification of a SPARC Family Polypeptide

A SPARC family polypeptide provided by the present invention may beproduced in a prokaryotic or eukaryotic cell using any known method, forexample, recombinant DNA techniques. Recombination techniques may beconducted as described herein below, or for example, by the methodsdisclosed in T. Maniatis et al., “Molecular Cloning”, 2nd Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N. T. (1989); NipponSeikagaku Kai (Biochemical Society of Japan) ed., “Zoku-Seikagaku JikkenKouza 1, Idenshi Kenkyuho II (Lectures on Biochemical Experiments(Second Series; 1), Methods for Gene Study II)”, Tokyo Kagaku Dojin,Japan (1986); Nippon Seikagaku Kai (Biochemical Society of Japan) ed.,“Shin-Seikagaku Jikken Kouza 2, Kakusan III (Kumikae DNA Gijutsu) (NewLectures on Biochemical Experiments 2, Nucleic Acids III (RecombinantDNA Technique))”, Tokyo Kagaku Dojin, Japan (1992); R. Wu (ed.),“Methods in Enzymology”, Vol. 68, Academic Press, New York (1980); R. Wuet al. (ed.), “Methods in Enzymology”, Vols. 100 & 101, Academic Press,New York (1983); R. Wu et al. (ed.), “Methods in Enzymology”, Vols. 153,154 & 155, Academic Press, New York (1987), etc. as well as bytechniques disclosed in the references cited therein, the disclosures ofwhich are hereby incorporated by reference. Such techniques and meansmay also be those which are individually modified/improved fromconventional techniques depending upon the object of the presentinvention.

A SPARC family polypeptide may be expressed and purified from arecombinant host cell. Recombinant host cells may be prokaryotic oreukaryotic, including but not limited to bacteria such as E. coli,fungal cells such as yeast, insect cells including but not limited todrosophila and silkworm derived cell lines, and mammalian cells and celllines.

In certain embodiments, when expressing and purifying a SPARC familypolypeptide of the present invention, techniques for improving proteinsolubility are employed to prevent the formation of inclusion body(which are insoluble fractions), and therefore obtaining largequantities of the polypeptide. SPARC accumulated in inclusion bodies isan inactive-type SPARC not retaining its physiological activities.

Solubility of a purified SPARC family polypeptide may be improved bymethods known in the art, and as described herein below.

For example, solubility may also be improved by expressing a functionalfragment, but not the full length SPARC family polypeptide. The fragmentexpressed should retain the sensitizing activity as described herein,albeit the activity may be lower than that of a full length polypeptide.

In one embodiment, a fragment containing one or two or three domains ofthe three conserved domains of the SPARC family members is expressed.

In addition, to increase the solubility of an expressed protein (e.g.,in E. coli), one can reduce the rate of protein synthesis by loweringthe growth temperature, using a weaker promoter, using a lower copynumber plasmid, lowering the inducer concentration, changing the growthmedium as described in Georgiou, G. & Valax, P. (1996, Current OpinionBiotechnol. 7, 190-197). This decreases the rate of protein synthesisand usually more soluble protein is obtained. One can also addprostethic groups or co-factors which are essential for proper foldingor for protein stability, or add buffer to control pH fluctuation in themedium during growth, or add 1% glucose to repress induction of the lacpromoter by lactose, which is present in most rich media (such as LB,2xYT). Polyols (e.g. sorbitol) and sucrose may also be added to themedia because the increase in osmotic pressure caused by these additionsleads to the accumulation of osmoprotectants in the cell, whichstabilize the native protein structure. Ethanol, low molecular weightthiols and disulfides, and NaCl may be added. In addition, chaperonesand/or foldases may be co-expressed with the desired polypeptide.Molecular chaperones promote the proper isomerization and cellulartargeting by transiently interacting with folding intermediates. Thebest characterized E. coli chaperone systems are: GroES-GroEL,DnaK-DnaJ-GrpE, ClpB. Foldases accelerate rate-limiting steps along thefolding pathway. Three types of foldases play an important role:peptidyl prolyl cis/trans isomerases (PPI's), disulfide oxidoreductase(DsbA) and disulfide isomerase (DsbC), protein disulfide isomerase (PDI)which is an eukaryotic protein that catalyzes both protein cysteineoxidation and disulfide bond isomerization. Co-expression of one or moreof these proteins with the target protein could lead to higher levels ofsoluble protein.

A SPARC family polypeptide of the present invention may be produced as afusion protein in order to improve its solubility and production. Thefusion protein comprises a SPARC family polypeptide and a secondpolypeptide fused together in frame. The second polypeptide may be afusion partner known in the art to improve the solubility of thepolypeptide to which it is fused, for example, NusA, bacterioferritin(BFR), GrpE, thioredoxin (TRX) and glutathione-S-transferase (GST).Madison, Wis.-based Novagen Inc. provides the pET 43.1 vector serieswhich permit the formation of a NusA-target fusion. DsbA and DsbC havealso shown positive effects on expression levels when used as a fusionpartner, therefore can be used to fuse with a SPARC polypeptide forachieving higher solubility.

In one embodiment, a SPARC family polypeptide is produced as a fusionpolypeptide comprising the SPARC family polypeptide and a fusion partnerthioredoxin, as described in U.S. Pat. No. 6,387,664, herebyincorporated by reference in its entirety. The thioredoxin-SPARC fusioncan be produced in E. coli as an easy-to-formulate, soluble protein in alarge quantity without losing the physiological activities of SPARC.Although U.S. Pat. No. 6,387,664 provides a fusion SPARC protein withSPARC fused to the C-terminus of thioredoxin, it is understood, for thepurpose of the present invention, a SPARC family polypeptide may befused either to the N-terminus or the C-terminus of a secondpolypeptide, so long as its sensitizing function is retained.

In addition to increase solubility, a fusion protein comprising a SPARCfamily polypeptide may be constructed for the easy detection of theexpression of the SPARC family polypeptide in a cell. In one embodiment,the second polypeptide which fused to the SPARC family polypeptide is areporter polypeptide. The reporter polypeptide, when served for suchdetection purpose, does not have to be fused with the SPARC familypolypeptide. It may be encoded by the same polynucleotide (e.g., avector) which also encodes the SPARC family polypeptide and beco-introduced and co-expressed in a target cell.

Preferably, the reporter polypeptide used in the invention is anautofluorescent protein (e.g., GFP, EGFP). Autofluorescent proteinsprovide a ready assay for identification of expression of apolynucleotide (and the polypeptide product) of interest. Because theactivity of the reporter polypeptide (and by inference its expressionlevel) can be monitored quantitatively using a flow sorter, it is simpleto assay many independent transfectants either sequentially or in bulkpopulation. Cells with the best expression can then be screened for orselected from the population. This is useful when selecting arecombinant cell comprising a SPARC family polypeptide or polynucleotidefor sensitizing treatment according to the present invention.Quantitative parameters such as mean fluorescence intensity and variancecan be determined from the fluorescence intensity profile of the cellpopulation (Shapiro, H., 1995, Practical Flow Cytometry, 217-228).Non-limiting examples of reporter molecules useful in the inventioninclude luciferase (from firefly or other species), chloramphenicolacetyltransferase, β-galactosidase, green fluorescent protein (GFP),enhanced green fluorescent protein (EGFP), and dsRed.

Expression of the SPARC polypeptide (either by itself, or as a fusionprotein) can also be directly determined by an immunoassay such as anELISA (enzyme-linked immunoabsorbent assay) (see e.g., U.S. Pat. No.5,962,320; U.S. Pat. No. 6,187,307; U.S. Pat. No. 6,194,205), westernblot, or by other methods routine in the art. The expression of a SPARCfamily polypeptide can be indirectly detected by detecting thetranscript of the protein (e.g., by hybridization analysis such asNorthern blot or DNA Microarray, or by PCR).

In one embodiment, a polynucleotide encoding a second polypeptide isfused to a polynucleotide encoding a SPARC family polypeptide through anintervening linker sequence which encodes for a linker polypeptide.

In another embodiment, the linker polypeptide comprises a proteasecleavage site comprising a peptide bond which is hydrolyzable by aprotease. As a result, the SPARC family polypeptide can be separatedfrom the second polypeptide after expression by proteolysis. The linkercan comprise one or more additional amino acids on either side of thebond to which the catalytic site of the protease also binds (see, e.g.,Schecter and Berger, 1967, Biochem. Biophys. Res. Commun. 27, 157-62).Alternatively, the cleavage site of the linker can be separate from therecognition site of the protease and the two cleavage site andrecognition site can be separated by one or more (e.g., two to four)amino acids. In one aspect, the linker comprises at least 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50 or more amino acids. More preferably thelinker is between 5 and 25 amino acids in length, and most preferably,the linker is between 8 and 15 amino acids in length.

Some proteases useful according to the invention are discussed in thefollowing references: V. Y. H. Hook, Proteolytic and cellular mechanismsin prohormone and proprotein processing, RG Landes Company, Austin,Tex., USA (1998); N. M. Hooper et al., 1997, Biochem. J. 321:265-279;Werb, 1997, Cell 91: 439-442; Wolfsberg et al., 1995, J. Cell Biol. 131:275-278; Murakami and Etlinger, 1987, Biochem. Biophys. Res. Comm. 146:1249-1259; Berg et al., 1995, Biochem. J. 307: 313-326; Smyth andTrapani, 1995, Immunology Today 16: 202-206; Talanian et al., 1997, J.Biol. Chem. 272: 9677-9682; and Thornberry et al., 1997, J. Biol. Chem.272: 17907-17911. Suitable proteases include, but are not limited to,those listed in Table 1 below.

TABLE 1 Proteases and Their Cleavage Signals Cleavage Signal (ExemplaryLinker Protease Polynucleotide Sequence) subtilisn/kexin family RXKR(furin, PC1, PC2, PC4, (SEQ ID NO. 49) PACE4, PC5, PC) (CGC NNN AAG CGC)(SEQ ID NO. 50) MMP-2 PLGLWA (SEQ ID NO. 51) (CCC CTG GGC CTG TGG GCC)(SEQ ID NO. 52) MT1-MMP PLGLWA (SEQ ID NO. 51) (CCC CTG GGC CTG TGG GCC)(SEQ ID NO. 52) Protease Cleavage Signal-Amino Acid Sequence (ExemplaryLinker Polynucleotide Sequence) caspase-1 YEVDGW (SEQ ID NO. 53) (TACGAG GTG GAC GGC TGG) (SEQ ID NO. 54) caspase-2 VDVADGW (SEQ ID NO. 55)(GTG GAC GTG GCC GAC GGC TGG) (SEQ ID NO. 56) caspase-3 VDQMDGW (SEQ IDNO. 57) (GTG GAC CAG ATG GAC GGC TGG) (SEQ ID NO. 58) caspase-4 LEVDGW(SEQ ID NO. 59) CTG GAG GTG GAC GGC TGG) (SEQ ID NO. 60) caspase-6VQVDGW (SEQ ID NO. 61) (GTG CAG GTG GAC GGC TGG) (SEQ ID NO. 62)caspase-7 VDQVDGW (SEQ ID NO. 63) (GTG GAC CAG GTG GAC GGC TGG) (SEQ IDNO. 64) caspase-8 DXXD (SEQ ID NO. 65) (GAC NNN NNN GAC) (SEQ ID NO. 66)caspase-9 DXXD (SEQ ID NO. 65) (GAC NNN NNN GAC) alpha-secretase amyloidprecursor protein (APP) proprotein convertase RGLT (subtilisin/kexin(SEQ ID NO. 67) isozyme SKI-1 (CGC GGC CTG ACC) (SEQ ID NO. 68)proprotein convertases cleavage at hydrophobic residues (e.g., Leu, Phe,Val, or Met) or at small acid residues such as Ala or Thr foot and mouthdisease NFDLLKLAGDVESNPGP virus, protease 2A (SEQ ID NO. 69) (AAC TTCGAC CTG CTG AAG CTG GCC GGC GAC GTG GAG AGC AAC CCC GGC CCC) (SEQ ID NO.70) signal peptidase A-X-A-X (SEQ ID NO. 71) (GCC NNN GCC NNN) (SEQ IDNO. 72) aminopeptidases (e.g., LTK arginine aminopeptidase, (SEQ ID NO.73) lysine aminopeptidase, (CTG ACC AAG) aminopeptidase B, (SEQ ID NO.74) trypsin) insulin degrading enzyme GGFLRKVGQ (SEQ ID NO. 75) (GGC GGCTTC CTG CGC AAG GTG GGC CAG) (SEQ ID NO. 76)

Additional linker polypeptides can be obtained from the substrates forproopiomelanocortin converting enzyme (PCE); chromaffin granule asparticprotease (CGAP); prohormone thiol protease; carboxypeptidases (e.g.,carboxypeptidase E/H, carboxypeptidase D and carboxypeptidase Z); prolylendopeptidase; and high molecular weight protease.

Cell surface proteases also can be used with cleavable linkers accordingto the invention and include, but are not limited to: Aminopeptidase N;Puromycin sensitive aminopeptidase; Angiotensin converting enzyme;Pyroglutamyl peptidase II; Dipeptidyl peptidase IV; N-arginine dibasicconvertase; Endopeptidase 24.15; Endopeptidase 24.16; Amyloid precursorprotein secretases alpha, beta and gamma; Angiotensin converting enzymesecretase; TGF alpha secretase; TNF alpha secretase; FAS ligandsecretase; TNF receptor-I and -II secretases; CD30 secretase; KL1 andKL2 secretases; IL6 receptor secretase; CD43, CD44 secretase; CD16-I andCD16-II secretases; L-selectin secretase; Folate receptor secretase; MMP1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15; Urokinase plasminogenactivator; Tissue plasminogen activator; Plasmin; Thrombin; BMP-1(procollagen C-peptidase); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11;and, Granzymes A, B, C, D, E, F, G, and H.

An alternative to relying on cell-associated proteases is to use a aself-cleaving linker. For example, the foot and mouth disease virus(FMDV) 2A protease may be used as a linker. This is a short polypeptideof 17 amino acids that cleaves the polyprotein of FMDV at the 2A/2Bjunction. The sequence of the FMDV 2A propeptide is NFDLLKLAGDVESNPGP(SEQ ID NO. 77). Cleavage occurs at the C-terminus of the peptide at thefinal glycine-proline amino acid pair and is independent of the presenceof other FMDV sequences and cleaves even in the presence of heterologoussequences.

Insertion of this sequence between two protein coding regions (i.e.,between the SPARC family polypeptide and the second polypeptide of afusion protein according to the invention) results in the formation of aself-cleaving chimera which cleaves itself into a C-terminal fragmentwhich carries the C-terminal proline of the 2A protease on itsN-terminal end, and an N-terminal fragment that carries the rest of the2A protease peptide on its C-terminus (see, e.g., P. deFelipe et al.,Gene Therapy 6: 198-208 (1999)). Self-cleaving linkers and additionalprotease-linker combinations are described further in WO 0120989, theentirety of which is incorporated by reference herein.

Polynucleotides encoding linker sequences described above can be clonedfrom sequences encoding the natural substrates of an appropriateprotease or can be chemically synthesized using methods routine in theart.

When expressing a SPARC family polypeptide in a human cell, e.g., invitro or in vivo, the codons selected for such the polynucleotideencoding the SPARC family polypeptide are preferably those which aremost frequently used in humans, such as those listed in Table 2 below.The exemplary polynucleotide sequences shown in Table 4 rely on codonswhich are most frequently used in humans.

TABLE 2 Preferred DNA Codons For Human Use Codons 3 1 Preferred AminoAcids Letter Code Letter Code in Human Genes Alanine Ala A GCC GCT GCAGCG Cystein Cys C TGT TGT Aspartic Acid Asp D GAC GAT Glutamic Acid GluE GAG GAA Phenylalanine Phe F TTC TTT Glycine Gly G GGC GGG GGA GGTHistidine His H CAC CAT Isoleucine Ile I ATC ATT ATA Lysine Lys K AAGAAA Leucine Leu L CTG TTG CTT CTA TTA Methionine Met M ATG AsparagineAsn N AAC AAT Proline Pro P CCC CCT CCA CCG Glutamine Gln Q CAG CAAArginine Arg R CGC AGG CGG AGA CGA CGT Serine Ser S AGC TCC TCT AGT TCATCG Threonine Thr T ACC ACA ACT ACG Valine Val V GTG GTC GTT GTATryprophan Trp W TGG Tyrosine Tyr Y TAC TAT

The uppermost codons represent those most preferred for use in humangenes. Underlined codons are almost never used in human genes and aretherefore not preferred.

In another embodiment, the present invention provides (a) apolynucleotide encoding the polypeptide of a SPARC family polypeptide ora fusion polypeptide comprising a SPARC family polypeptide; and (b) apolynucleotide hybridizing to the polynucleotide of (a) under astringent hybridization condition.

Techniques for polynucleotide manipulation to perform the aboveembodiments of the invention are well known in the art. (See, e.g.,Sambrook et al., 1989; Ausubel et al. 1987 and in Annual Reviews ofBiochemistry, 1992, 61:131-156). Reagents useful in applying suchtechniques, such as restriction enzymes and the like, are widely knownin the art and commercially available from a number of vendors.

Polynucleotide sequences for use in the present invention may also beproduced in part or in total by chemical synthesis, e.g. by thephosphoramidite method described by Beaucage, et al., 1981, Tetra.Letts., 22:1859-1862, or the triester method (Matteucci et al., 1981, J.Am. Chem. Soc., 103:3185), which may be performed on commercialautomated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single-stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing the strandtogether under appropriate conditions, or by synthesizing thecomplementary strand using DNA polymerase with an appropriate primersequence.

In one embodiment, the SPARC family polynucleotide provided by thepresent invention exists as a vector, preferably, an expression vector.

Expression Vectors

A polynucleotide coding for a SPARC family polypeptide sequence may beincorporated into vectors capable of introduction into and replicationin a prokaryotic or eukaryotic cell to produce a SPARC familypolypeptide of the present invention for sensitizing treatment, or itcan be used to transfect or infect a cell or tissue and achieve thesensitizing function directly by expressing the SPARC familypolypeptide. The vectors may or may not integrate within the genome ofthe transfected or infected cell.

Useful polynucleotide molecules encoding a SPARC family polypeptide ofthe present invention may be cloned into an expression vector beforethey are introduced into an appropriate cell and may be passage in cellsto generate useable quantities of these polynucleotides.

Suitable vectors for the invention may be plasmid or viral vectors.Plasmid expression vectors include, but are not limited to, plasmidsincluding pBR322, pUC, pcDNA3.1 or Bluescript™. Viral vectors include,but are not limited to baculoviruses, adenoviruses, poxviruses,adenoassociated viruses (AAV), and retrovirus vectors (Price et al,1987, Proc. Natl. Acad. Sci. USA, 84:156-160) such as the MMLV basedreplication incompetent vector pMV-7 (Kirschmeier et al., 1988, DNA,7:219-225), as well as human and yeast modified chromosomes (HACs andYACs).

The expression vectors may comprise one or more regulatory elements todrive and/or enhance expression of upstream or downstreampolynucleotides. These regulatory sequences are selected on the basis ofthe cells to be used for introduction and/or expression, and areoperatively linked to a polynucleotide sequence to be expressed. Theterm “regulatory elements” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory elements are described, for example, in Goeddel; 1990,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif.

Regulatory elements include those which direct expression of anucleotide sequence in many types of subject cells as well as thosewhich direct expression of the nucleotide sequence only in certainsubject cells (e.g., tissue-specific regulatory sequences).

Regulatory elements also include those which direct constitutiveexpression of an operatively linked polynucleotide sequence and thosewhich direct inducible expression of the polynucleotide sequence.

Preferably, suitable promoters may be used. For example, such promotersmay include tryptophan (trp) promoter, lactose (lac) promoter,tryptophan-lactose (tac) promoter, lipoprotein (lpp) promoter, λ phageP_(L) promoter, etc. in the case of plasmids where Escherichia coli isused as a host; and GAL1, GAL10 promoters, etc. in the case of plasmidswhere yeast is used as a host.

Some eukaryotic promoters and enhancers have a broad range of cells inwhich they can activate and/or modulate transcription while others arefunctional only in a limited subset of cell types (See e.g., Voss etal., 1986, Trends Biochem. Sci., 11:287; and Maniatis et al., supra, forreviews). For example, the SV40 early gene enhancer is very active in awide variety of cell types from many mammalian species and has beenwidely used for the expression of proteins in mammalian cells (Dijkemaet al., 1985, EMBO J. 4:761). Two other examples of promoter/enhancerelements active in a broad range of mammalian cell types are those fromthe human elongation factor 1α gene (Uetsuki et al., 1989, J. Biol.Chem., 264:5791; Kim et al., 1990, Gene, 91:217; and Mizushima, et al.,1990, Nagata, Nuc. Acids. Res., 18:5322) and the long terminal repeatsof the Rous sarcoma virus (Gorman et al., 1982, Proc. Natl. Acad. Sci.USA, 79:6777) and the human cytomegalovirus (Boshart et al., 1985, Cell,41:521).

Suitable promoters for eukaryotic cell expression include, but are notlimited to, TRAP promoters, adenoviral promoters, such as the adenoviralmajor late promoter; the cytomegalovirus (CMV) promoter; the respiratorysyncytial virus (RSV) promoter; inducible promoters, such as the MMTpromoter, the metallothionein promoter, heat shock promoters; thealbumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; ITRs; the β-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter thatcontrols the polynucleotide encoding the polypeptide and the sequencesof native promoters may be found in the art (see Agrawal et al., 2000,J. Hematother. Stem Cell Res., 795-812; Cournoyer et al., 1993, Annu.Rev. Immunol., 11:297-329; van de Stolpe et al., 1996, J. Mol. Med.,74:13-33; Herrmann, 1995, J. Mol. Med., 73:157-63)

A variety of enhancer sequences can be used in the instant inventionincluding but not limited to: Immunoglobulin Heavy Chain enhancer;Immunoglobulin Light Chain enhancer; T-Cell Receptor enhancer; HLA DQ αand DQ β enhancers; β-Interferon enhancer; interleukin-2 enhancer;Interleukin-2 Receptor enhancer; MHC Class II 5_(a) ^(k) enhancer; MHCClass II HLA-DRαenhancer; β-Actin enhancer; Muscle Creatine Kinaseenhancer; Prealbumin (Transthyretin) enhancer; Elastase I enhancer;Metallothionein enhancer; Collagenase enhancer; Albumin Gene enhancer;α-Fetoprotein enhancer; β-Globin enhancer; c-fos enhancer; c-HA-rasenhancer; Insulin enhancer; Neural Cell Adhesion Molecule (NCAM)enhancer; α₁-Antitrypsin enhancer; H2B (TH2B) Histone enhancer; Mouse orType I Collagen enhancer; Glucose-Regulated Proteins (GRP94 and GRP78)enhancer; Rat Growth Hormone enhancer; Human Serum Amyloid A (SAA)enhancer; Troponin I (TN I) enhancer; Platelet-Derived Growth Factorenhancer; Duchenne Muscular Dystrophy enhancer; SV40 Polyoma enhancer;Retrovirusal enhancer; Papilloma Virus enhancer; Hepatitis B Virusenhancer; Human Immunodeficiency enhancer; Cytomegalovirus enhancer; andGibbon Ape Leukemia Virus enhancer.

Exemplary inducible promoter/enhancer sequences and their inducers arelisted below.

TABLE 3 Useful Inducible Promoters/Enhancers Element Inducer MTIIPhorbolEster(TFA) Heavymetals MMTV (mouse mammary tumor virus)Glucocorticoids β-Interferon poly(rI) × poly(rc) Adenovirus 5 E2 Elac-jun Phorbol Ester (TPA), H₂O₂ Collagenase Phorbol Ester (TPA)Stromelysin Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TFA) Murine MXGene Interferon, Newcastle Disease Virus GRP78 Gene A23187α-2-Macroglobulin IL-6 Vimentin Serum MHC ClassI Gene H-2kB InterferonHSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester (TPA) TumorNecrosis Factor FMA Thyroid Stimulating Hormone α Gene Thyroid HormoneAdditional Eukaryotic regulatory sequences may be obtained from theEukaryotic Promoter Data Base EPDB) also can be used to drive expressionof a polynucleotide.

In certain embodiments of the invention, the delivery of a vector in acell may be identified in vitro or in vivo by including a selectionmarker in the expression construct, such as described herein above. Themarker would result in an identifiable change to the modified cellpermitting easy identification of expression. Usually the inclusion of adrug selection marker aids in cloning and in the selection oftransformants. Genes which can be used as selectable markers inEukaryotic cells are known in the art and include, examples of dominantselectable markers include the bacterial aminoglycoside 3′phosphotransferase gene (also referred to as the neo gene) which confersresistance to the drug G418 in mammalian cells, the bacterial hygromycinG phosphotransferase (hyg) gene which confers resistance to theantibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyltransferase gene (also referred to as the gpt gene) which confers theability to grow in the presence of mycophenolic acid. Other selectablemarkers are not dominant in that there use must be in conjunction with acell line that lacks the relevant enzyme activity. Examples ofnon-dominant selectable markers include the thymidine kinase (tk) genewhich is used in conjunction with tk⁻ cell lines, the CAD gene which isused in conjunction with CAD-deficient cells and the mammalianhypoxanthine-guanine phosphoribosyl transferase (hprt) gene which isused in conjunction with hprt⁻ cell lines. A review of the use ofselectable markers in mammalian cell lines is provided in Sambrook, J.et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, New York pp. 16.9-16.15.

Alternatively, genes encoding enzymes, such as herpes simplex virusthymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase(CAT) (prokaryotic) may be employed to provide selectable markers.Immunologic markers also can be employed. The exact type selectablemarker employed is not believed to be important, so long as it iscapable of being expressed simultaneously with the polynucleotideencoding a polypeptide of interest. Further examples of selectablemarkers are well known to one of skill in the art.

Where a cDNA insert is employed to express a SPARC family polypeptide ofthe invention, one typically will desire to include a polyadenylationsignal to effect proper polyadenylation of the polynucleotidetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. These elements can serve to enhance messagelevels and to minimize read through from the expression cassette intoother sequences.

Recombinant Cell Production: —Introducing a SPARC family PolynucleotideInto a Cell for Expression and/or Sensitization

As described above, a SPARC family polynucleotide of the invention maybe introduced into a cell in order to express the SPARC familypolypeptide for purification or to express and achieve the sensitizingeffect of the invention according to methods well known in the art, forexample, in Current Protocols in Molecular Biology, Ausubel, F. M. etal. (eds.), 1989, Greene Publishing Associates, Section 9.1 and inMolecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al.,1989, Cold Spring Harbor Laboratory Press.

Several non-viral methods for the transfer of vectors into culturedmammalian cells are contemplated by the present invention. These includecalcium phosphate precipitation (Graham, et al., 1973; Chen, et al.,1987; Rippe, et al., 1990) DEAE-dextran (Gopal, 1985), electroporation(Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection(Harland, et al., 1985), DNA-loaded liposomes (Nicolau, et al., 1982;Fraley et al., 1979) and lipofectamine-DNA complexes, cell sonication(Fechheimer et al., 1987), gene bombardment using high velocitymicroprojectiles (Yang et al., 1990), and receptor-mediated transfection(Wu, et al., 1987; Wu, et al., 1988). Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

Once the vector has been delivered into the cell, the polynucleotideencoding a SPARC family polypeptide may be positioned and expressed atdifferent sites. In certain embodiments, the polynucleotide encoding aSPARC family polypeptide may be stably integrated into the genome of thecell. This integration may be via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation), see Holmes-Son et al., 2001, Adv. Genet. 43: 33-69.In yet further embodiments, the polynucleotide encoding a SPARC familypolypeptide may be stably maintained in the cell as a separate, episomalsegment of DNA. Such polynucleotide segments or “episomes” encodesequences sufficient to permit maintenance and replication independentof or in synchronization with the subject cell cycle. How the expressionconstruct is delivered to a cell and where in the cell thepolynucleotide remains is well known in the art and is dependent on thetype of expression construct employed.

Cell lines derived from mammalian species which may be suitable fortransfection and infection of a SPARC family polynucleotide and forexpression and purification of a recombinant polypeptide, may becommercially available. These cell lines include but are not limited to,CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCCCCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL171), L-cells, HEK-293 (ATCC CRL1573), NS0 (ECACC85110503) and HT1080.

Cell cultures may be prepared in various ways for gene transfer invitro. In order for the cells to be kept viable while in vitro and incontact with the expression construct, it is necessary to ensure thatthe cells maintain contact with the correct ratio of oxygen and carbondioxide and nutrients but are protected from microbial contamination.

Transfer of the construct may be performed by any of the methods knownin the art and as described herein below. Some methods may beparticularly applicable for transfer in vitro but it may be applied toin vivo use as well.

Transfection Mediated by CaPO₄

A polynucleotide encoding a SPARC family polypeptide can be introducedinto cells by forming a precipitate containing the polynucleotide andcalcium phosphate. For example, a HEPES-buffered saline solution can bemixed with a solution containing calcium chloride and polynucleotide toform a precipitate and the precipitate is then incubated with cells. Aglycerol or dimethyl sulfoxide shock step can be added to increase theamount of polynucleotide taken up by certain cells. CaPO₄-mediatedtransfection can be used to stably (or transiently) transfect cells andis only applicable to in vitro modification of cells. Protocols forCaPO₄-mediated transfection can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (eds.), 1989, Greene PublishingAssociates, Section 9.1 and in Molecular Cloning: A Laboratory Manual,2nd Edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory PressSections 16.32-16.40 or other standard laboratory manuals.

Dubensky et al. (1984) successfully injected polyomavirus DNA in theform of CaPO₄ precipitates into liver and spleen of adult and newbornmice demonstrating active viral replication and acute infection.Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of CaPO₄ precipitated plasmids results inexpression of the transfected genes. Thus the polynucleotide encoding aSPARC family polypeptide may also be transferred in a similar manner invivo to express a desired SPARC family polypeptide as described above.

Transfection Mediated by DEAE-Dextran

A polynucleotide encoding a SPARC family polypeptide can be introducedinto cells by forming a mixture of the polynucleotide and DEAE-dextranand incubating the mixture with the cells. A dimethylsulfoxide orchloroquine shock step can be added to increase the amount ofpolynucleotide uptake. Protocols for DEAE-dextran-mediated transfectioncan be found in Current Protocols in Molecular Biology, Ausubel, F. M.et al. (eds.), 1989, Greene Publishing Associates, Section 9.2 and inMolecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al.,1989, Cold Spring Harbor Laboratory Press, Sections 16.41-16.46 or otherstandard laboratory manuals.

Electroporation

A polynucleotide encoding a SPARC family polypeptide can also beintroduced into cells by incubating the cells and the polynucleotidetogether in an appropriate buffer and subjecting the cells to ahigh-voltage electric pulse. The efficiency with which polynucleotide isintroduced into cells by electroporation is influenced by the strengthof the applied field, the length of the electric pulse, the temperature,the conformation and concentration of the polynucleotide and the ioniccomposition of the media. Electroporation can be used to stably (ortransiently) transfect a wide variety of cell types. Protocols forelectroporating cells can be found in Ausubel, F. M. et al. (eds.),supra, Section 9.3 and in Sambrook et al., supra, Sections 16.54-16.55or other standard laboratory manuals.

Liposome-Mediated Transfection (“Lipofection”)

A polynucleotide encoding a SPARC family polypeptide also can beintroduced into cells by mixing the polynucleotide with a liposomesuspension containing cationic lipids. The polynucleotide/liposomecomplex is then incubated with cells. Liposome mediated transfection canbe used to stably (or transiently) transfect cells in culture in vitro.Protocols can be found in Ausubel, F. M. et al. (eds.), supra, Section9.4 and other standard laboratory manuals. Additionally, gene deliveryin vivo has been accomplished using liposomes. See for example Nicolauet al., 1987, Meth. Enz., 149:157-176; Wang, et al., 1987, Proc. Natl.Acad. Sci. USA, 84:7851-7855; Brigham et al., 1989, Am. J. Med. Sci.,298:278; and Gould-Fogerite et al., 1989, Gene, 84:429-438.

Direct Injection

A polynucleotide encoding a SPARC family polypeptide can be introducedinto cells by directly injecting the polynucleotide into the cells. Foran in vitro culture of cells, polynucleotide can be introduced bymicroinjection. Since each cell is microinjected individually, thisapproach is very labor intensive when modifying large numbers of cells.However, a situation where microinjection is a method of choice is inthe production of transgenic animals (discussed in greater detailbelow). In this situation, the polynucleotide is stably introduced intoa fertilized oocyte which is then allowed to develop into an animal. Theresultant animal contains cells carrying the polynucleotide introducedinto the oocyte. Direct injection may be used to introduce thepolynucleotide encoding a SPARC family polypeptide into cells in vivo(see e.g., Acsadi et al., 1991, Nature, 332: 815-818; Wolff et al.,1990, Science, 247:1465-1468). A delivery apparatus (e.g., a “gene gun”)for injecting DNA into cells in vivo can be used. Such an apparatus iscommercially available (e.g., from BioRad).

Receptor-Mediated DNA Uptake

A polynucleotide encoding a SPARC family polypeptide also can beintroduced into cells by complexing the polynucleotide to a cation, suchas polylysine, which is coupled to a ligand for a cell-surface receptor(see for example Wu, et al., 1988, J. Biol. Chem., 263:14621; Wilson etal., 1992, J. Biol. Chem., 267:963-967; and U.S. Pat. No. 5,166,320).Binding of the polynucleotide-ligand complex to the receptor facilitatesuptake of the polynucleotide by receptor-mediated endocytosis. Receptorsto which a polynucleotide-ligand complex have targeted include thetransferrin receptor and the asialoglycoprotein receptor. Apolynucleotide-ligand complex linked to adenovirus capsids whichnaturally disrupt endosomes, thereby releasing material into thecytoplasm can be used to avoid degradation of the complex byintracellular lysosomes (see for example Curiel et al., 1991, Proc.Natl. Acad. Sci. USA, 88:8850; Cristiano et al., 1993, Proc. Natl. Acad.Sci. USA, 90:2122-2126). Receptor-mediated polynucleotide uptake can beused to introduce the polynucleotide encoding a SPARC family polypeptideinto cells either in vitro or in vivo and, additionally, has the addedfeature that polynucleotide can be selectively targeted to a particularcell type by use of a ligand which binds to a receptor selectivelyexpressed on a target cell of interest.

Viral-Mediated Gene Transfer

Another approach for introducing a polynucleotide encoding a SPARCfamily polypeptide into a cell is by use of a viral vector containingthe polynucleotide encoding a SPARC family polypeptide. Infection ofcells with a viral vector has the advantage that a large proportion ofcells receive the polynucleotide, which can obviate the need forselection of cells which have received the polynucleotide. Additionally,molecules encoded within the viral vector, e.g., by a cDNA contained inthe viral vector, are expressed efficiently in cells which have taken upviral vector polynucleotide and viral vector systems can be used eitherin vitro or in vivo.

Nonreplicating viral vectors can be produced in packaging cell lineswhich produce virus particles which are infectious butreplication-defective, rendering them useful vectors for introduction ofpolynucleotide into a cell lacking complementary genetic informationenabling encapsidation (Mann et al., 1983, cell, 33:153; Miller andButtimore, Mol. Cell. Biol., 1986, 6:2895 (PA317, ATCC CRL9078).Packaging cell lines which contain amphotrophic packaging genes able totransform cells of human and other species origin are preferred.

Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990,in Fields et al., Ceds, Virology, Raven Press, New York, pp. 1437-1500).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene, functions as a signal for packaging of the genome intovirions. Two long terminal repeat (LTR) sequences are present at the 5′and 3′ ends of the viral genome. These contain strong promoter andenhancer sequences and are also required for integration in the subjectcell genome (Coffin, supra).

Defective retroviruses are well characterized for use in gene transferfor gene therapy purposes (for a review see Miller, 1990, Blood 76:271).

Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.), 1989, GreenePublishing Associates, Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are well known to those skilled in the art. Examples ofsuitable packaging virus lines include Crip, Cre, 2 and Am. Retroviruseshave been used to introduce a variety of genes into many different celltypes, including epithelial cells, endothelial cells, lymphocytes,myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (seefor example Eglitis, et al., 1985, Science, 230:1395-1398; Danos, etal., 1988, Proc. Nail. Acad. Sci. USA, 85:6460-6464; Wilson et al.,1988, Proc. Natl. Acad. Sci. USA, 85:3014-3018; Armentano et al., 1990,Proc. Natl. Acad. Sci. USA, 87:6141-6145; Huber et al., 1991, Proc.Natl. Acad. Sci. USA, 88:8039-8043; Ferry et al., 1991, Proc. Natl.Acad. Sci. USA, 88:8377-8381; Chowdhury et al., 1991, Science,254:1802-1805; van Beusechem et al., 1992, Proc. Natl. Acad. Sci. USA,89:7640-7644; Kay et al., 1992, Human Gene Therapy, 3:641-647; Dai etal., 1992, Proc. Natl. Acad. Sci. USA, 89:10892-10895; Hwu et al., 1993,J. Immunol., 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573). Retroviralvectors require target cell division in order for the retroviral genome(and foreign polynucleotide inserted into it) to be integrated into thesubject genome to stably introduce polynucleotide into the cell. Thus,it may be necessary to stimulate replication of the target cell.

Adenovirus

Knowledge of the genetic organization of adenovirus, a 36 kB, linear anddouble-stranded DNA virus, allows substitution of a large piece ofadenoviral DNA with foreign sequences up to 7 kB (Grunhaus, et al.,1992, Seminar in Virology, 3:237-252). In contrast to retrovirus, theinfection of adenoviral DNA into subject cells does not result inchromosomal integration because adenoviral DNA can replicate in anepisomal manner without potential genotoxicity. Also, adenoviruses arestructurally stable, and no genome rearrangement has been detected afterextensive amplification. Adenovirus can infect virtually all epithelialcells regardless of their cell cycle stage.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range, and high infectivity. Both ends of the viral genomecontain 100-200 base pair (bp) inverted terminal repeats (ITR), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression, and host cellshut off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5′ tripartite leader (TL) sequence which makes them preferredmRNAs for translation.

The genome of an adenovirus can be manipulated such that it encodes andexpresses a gene product of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See for exampleBerkner, et al., 1988, BioTechniques, 6:616; Rosenfeld, et al., 1991,Science, 252:431-434; and Rosenfeld, et al., 1992, Cell, 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) arewell known to those skilled in the art. Recombinant adenoviruses areadvantageous in that they do not require dividing cells to be effectivegene delivery vehicles and can be used to infect a wide variety of celltypes, including airway epithelium (Rosenfeld, et al., 1992, citedsupra), endothelial cells (Lemarchand, et al., 1992, Proc. Natl. Acad.Sci. USA, 89:6482-6486), hepatocytes (Herz, et al., 1993, Proc. Natl.Acad. Sci. USA, 90:2812-2816) and muscle cells (Quantin, et al., 1992,Proc. Natl. Acad. Sci. USA, 89:2581-2584). Additionally, introducedadenoviral polynucleotide (and foreign DNA contained therein) is notintegrated into the genome of a subject cell but remains episomal,thereby avoiding potential problems that can occur as a result ofinsertional mutagenesis in situations where introduced polynucleotidebecomes integrated into the subject genome (e.g., retroviral DNA).Moreover, the carrying capacity of the adenoviral genome for foreign DNAis large (up to 8 kilobases) relative to other gene delivery vectors(Berkner, et al. cited supra; Haj-Ahmand, et al., 1986, J. Virol.,57:267). Most replication-defective adenoviral vectors currently in useare deleted for all or parts of the viral E1 and E3 genes but retain asmuch as 80% of the adenoviral genetic material.

Recombinant adenovirus may be generated by methods known in the art,e.g., as described in U.S. Pat. No. 6,194,191, incorporated herein byreference.

Generation and propagation of the adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham, et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones, et al., 1978), the current adenovirus vectors, with the help of293 cells, carry foreign DNA in either the E1, the E3 or both regions(Graham, et al., 1991). In nature, adenovirus can package approximately105% of the wild-type genome (Ghosh-Choudhury, et al., 1987), providingcapacity for about 2 extra kB of DNA. Combined with the approximately5.5 kB of DNA that is replaceable in the E1 and E3 regions, the maximumcapacity of the current adenovirus vector is under 7.5 kB, or about 15%of the total length of the vector. More than 80% of the adenovirus viralgenome remains in the vector backbone and is the source of vector-bornecytotoxicity. Also, the replication deficiency of the E1 deleted virusis incomplete. For example, leakage of viral gene expression has beenobserved with the currently available adenovirus vectors at highmultiplicities of infection (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in themethod of the present invention. This is because Adenovirus type 5 is ahuman adenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication-defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding a polypeptide of interest at the position from which the E1coding sequences have been removed. However, the position of insertionof the coding region of a selected polynucleotide within the adenovirussequences is not critical to the present invention.

Adenovirus is easy to grow and manipulate and exhibits broad subjectrange in vitro and in vivo. This group of viruses can be obtained inhigh titers, e.g., 10⁹-10¹¹ plaque-forming unit (PFU)/ml, and they arehighly infective. The life cycle of adenovirus does not requireintegration into the subject cell genome.

Adenovirus vectors have been used in eukaryotic gene expression(Levrero, et al., 1991, Gene, 101:195-202; Gomez-Foix, et al., 1992, J.Biol. Chem., 267:25129-25134) and vaccine development (Grunhaus, et al.,1992, Seminar in Virology, 3:237-252; Graham, et al., 1992,Biotechnology, 20:363-390). Animal studies suggested that recombinantadenovirus could be used for gene therapy (Stratford-Perricaudet, etal., 1991, in: Human Gene Transfer, O. Cohen-Haguenauer, Ceds), JohnLibbey Eurotext, France; Stratford-Perricaudet, et al., 1990, Hum. GeneTher., 1:241-256; Rich, et al., 1993, Nature, 361:647-650). Experimentsin administering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld, et al., 1991, Science, 252:431-434;Rosenfeld, et al., 1992, Cell, 68:143-155), muscle injection (Ragot, etal., 1993, Nature, 361:647-650), peripheral intravenous injection (Herz,et al., 1993, Proc. Nat'l. Acad. Sci. USA 90:2812-2816), andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993,Science, 259:988-990).

Other Viral Vectors as Expression Vectors

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988, in: Rodriguez R L, Denhardt D T, ed. Vectors: A Surveyof Molecular Cloning Vectors and Their Uses. Stoneham: Butterworth, pp.467-492; Baichwal, et al., 1986 In: Kucherlapati R, ed. Gene Transfer.New York: Plenum Press, pp. 117-148; Coupar, et al., 1988, Gene,68:1-10), adeno-associated virus (AAV) (Baichwal, et al., 1986, supra;Hermonat, et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6466-6470) andherpesviruses may be employed. They offer several attractive featuresfor various mammalian cells (Friedmann, 1989, Science, 244:1275-1281;Baichwal, et al., 1986, supra; Coupar, et al., 1988, supra; Horwich, etal., 1990, J. Virol., 64:642-650).

Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal., 1992, Curr. Topics in Micro. and Immunol., 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see, forexample, Flotte et al., 1992, Am. J. Respir. Cell. Mol. Biol.,7:349-356; Samulski et al., 1989, J. Virol., 63:3822-3828; andMcLaughlin et al., 1989, J. Virol., 62:1963-1973). Vectors containing aslittle as 300 base pairs of AAV can be packaged and can integrate. Spacefor exogenous polynucleotide is limited to about 4.5 kb. An AAV vectorsuch as that described in Tratschin et al., 1985, Mol. Cell. Biol.,5:3251-3260 can be used to introduce polynucleotide into cells. Avariety of polynucleotides have been introduced into different celltypes using AAV vectors (see for example Hermonat, et al., 1984, Proc.Natl. Acad. Sci. USA, 81:6466-6470; Tratschin, et al., 1985, Mol. Cell.Biol., 4:2072-2081; Wondisford, et al., 1988, Mol. Endocrinol., 2:32-39;Tratschin, et al., 1984, J. Virol., 51:611-619; and Flotte, et al.,1993, J. Biol. Chem., 268:3781-3790).

After the transfer of a polynucleotide encoding a SPARC familypolypeptide into cells, the cells may be selected and used forsensitizing treatment according to the present invention. The efficacyof a particular expression vector system and method of introducingpolynucleotide into a cell can be assessed by standard approachesroutinely used in the art. For example, polynucleotide introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced polynucleotidecan be detected, for example, by Northern blotting, RNase protection orreverse transcriptase-polymerase chain reaction (RT-PCR). The geneproduct can be detected by an appropriate assay, for example byimmunological detection of a produced protein, such as with a specificantibody, or by a functional assay to detect a functional activity ofthe gene product, such as an enzymatic assay. Alternatively, anexpression system can first be optimized using a reporter gene linked tothe regulatory elements and vector to be used as described herein above.The reporter gene encodes a separate gene product which is easilydetectable and, thus, can be used to evaluate the efficacy of thesystem.

Since a SPARC family polypeptide is a secreted protein, itsextracellular level may also be detected to determine its expressionusing methods known in the art, such as Immunoblotting or Elisa.

Another way of increase the expression of a SPARC polynucleotide orpolypeptide in a cell is by endogenous gene activation, i.e., insertinga strong promoter before the natural SPARC family gene sequence in thegenome of the cell. Endogenous gene activation is a method ofintroducing, by homologous recombination with genomic DNA, DNA sequences(e.g., strong promoters) which are not normally functionally linked tothe endogenous gene and (1) which, when inserted into the host genome ator near the endogenous gene, serve to alter (e.g., activate) theexpression of the endogenous gene, and further (2) allow for selectionof cells in which the activated endogenous gene is amplified. Expressionof proteins by endogenous gene activation is well known in the art andis disclosed, for example in U.S. Pat. Nos. 5,733,761, 5,641,670, and5,733,746, and international patent publication Nos. WO 93/09222, WO94/12650, WO 95/31560, WO 90/11354, WO 91/06667 and WO 91/09955, thecontents of each of which are incorporated herein by reference in itsentirety.

In one embodiment, a endogenous SPARC family gene expression isactivated (e.g., increased) by inserting a tetracycline-inducibletetracycline promoter/operator to control its expression.

The methods described above to transfer polynucleotide into cells and tomake recombinant cells of the invention are merely for purposes ofillustration and are typical of those that might be used. However, otherprocedures may also be employed to obtain expression of a SPARC familypolypeptide in cells, as is understood in the art.

Cancer Therapy

Cancer is typically treated by surgery, chemotherapy or radiationtherapy. Biological therapies such as immunotherapy and gene therapy arealso being developed. Other therapies include hyperthermic therapy,photodynamic therapy, etc. (see National Cancer Institute home page atworld wide web nci.nih.gov).

Chemotherapy

Chemotherapy is the use of anti-cancer (cytotoxic) drugs to destroycancer cells. There are over 50 different chemotherapy drugs and someare given on their own, but often several drugs may be combined (this isknown as combination chemotherapy). An example list of chemotherapyagents, as described at World wide webcancerbacup.org.uk/info/actinomycin.htm, include: Actinomycin D,Adriamycin, Altretamine, Asparaginase, Bleomycin, Busulphan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, CPT-11,Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Doxorubicin,Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, fosfamide, Irinotecan, Liposomal Doxorubicin,Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin,Mitozantrone, Oxaliplatin, Procarbazine, Steroids, Streptozocin, Taxol,Taxotere, Taxotere—the TACT trial, Tamozolomide, Thioguanine, Thiotepa,Tomudex, Topotecan, Treosulfan, UFT (Uracil-tegufur), Vinblastine,Vincristine, Vindesine, Vinorelbine.

Because cancer cells may grow and divide more rapidly than normal cells,many anticancer drugs are made to kill growing cells. But certainnormal, healthy cells also multiply quickly, and chemotherapy can affectthese cells, too. This damage to normal cells causes side effects. Thefast-growing, normal cells most likely to be affected are blood cellsforming in the bone marrow and cells in the digestive tract (mouth,stomach, intestines, esophagus), reproductive system (sexual organs),and hair follicles. Some anticancer drugs may affect cells of vitalorgans, such as the heart, kidney, bladder, lungs, and nervous system.

The kinds of side effects one has and how severe they are depend on thetype and dose of chemotherapy one gets and how its body reacts. Sideeffects of chemotherapy include fatigue, nausea and vomiting, pain, hairloss, anemia, central nervous system problems, infection, blood clottingproblems, mouth, gum, and throat problems, diarrhea, constipation, nerveand muscle effects, effects on skin and nails, radiation recall, kidneyand bladder effects, flu-like symptoms, and fluid retention.

Radiation Therapy

One type of radiation therapy commonly used involves photons, “packets”of energy. X-rays were the first form of photon radiation to be used totreat cancer. Depending on the amount of energy they possess, the rayscan be used to destroy cancer cells on the surface of or deeper in thebody. The higher the energy of the x-ray beam, the deeper the x-rays cango into the target tissue. Linear accelerators and betatrons aremachines that produce x-rays of increasingly greater energy. The use ofmachines to focus radiation (such as x-rays) on a cancer site is calledexternal beam radiotherapy.

Gamma rays are another form of photons used in radiotherapy. Gamma raysare produced spontaneously as certain elements (such as radium, uranium,and cobalt 60) release radiation as they decompose, or decay. Eachelement decays at a specific rate and gives off energy in the form ofgamma rays and other particles. X-rays and gamma rays have the sameeffect on cancer cells.

Another technique for delivering radiation to cancer cells is to placeradioactive implants directly in a tumor or body cavity. This is calledinternal radiotherapy. (Brachytherapy, interstitial irradiation, andintracavitary irradiation are types of internal radiotherapy.) In thistreatment, the radiation dose is concentrated in a small area, and thepatient stays in the hospital for a few days. Internal radiotherapy isfrequently used for cancers of the tongue, uterus, and cervix.

Several new approaches to radiation therapy are being evaluated todetermine their effectiveness in treating cancer. One such technique isintraoperative irradiation, in which a large dose of external radiationis directed at the tumor and surrounding tissue during surgery.

Another investigational approach is particle beam radiation therapy.This type of therapy differs from photon radiotherapy in that itinvolves the use of fast-moving subatomic particles to treat localizedcancers. A sophisticated machine is needed to produce and accelerate theparticles required for this procedure. Some particles (neutrons, pions,and heavy ions) deposit more energy along the path they take throughtissue than do x-rays or gamma rays, thus causing more damage to thecells they hit. This type of radiation is often referred to as highlinear energy transfer (high LET) radiation.

Two types of investigational drugs are being studied for their effect oncells undergoing radiation. Radiosensitizers make the tumor cells morelikely to be damaged, and radioprotectors protect normal tissues fromthe effects of radiation. Hyperthermia, the use of heat, is also beingstudied for its effectiveness in sensitizing tissue to radiation.

Radioactive seed implants can be used as the sole treatment modality foradenocarcinoma of the prostate for appropriate patients with early stagedisease. The two most common sources are Iodine-125 and Palladium-103with no compelling clinical data that one is superior to the other. Theradioactive seed implant can be individually customized to a patient'sprostate to maximize the dose to the gland while minimizing the dose tothe surrounding normal structures. Prostate brachytherapy offers thehighest level of conformal radiation therapy for adenocarcinoma of theprostate. The prostate brachytherapy team at Thomas Jefferson Universityhas extensive experience in prostate brachytherapy and has presentedwork at national and international forums.

Prostate brachytherapy or radioactive seed implant is a highlytechnical, operator dependent method delivers the radiation energy byplacing many small radioactive seeds directly inside the prostate,effectively delivering the treatment “from the inside-out”. This is donein the operating room under general anesthesia, as a one-time procedure.These seeds can deliver high doses of radiation directly to the tumor,with little harm to the normal, healthy tissue around the prostate. Thismay be combined with 3-dimensional conformal radiation therapy in somesettings.

Other recent radiotherapy research has focused on the use ofradiolabeled antibodies to deliver doses of radiation directly to thecancer site (radioimmunotherapy). Antibodies are highly specificproteins that are made by the body in response to the presence ofantigens (substances recognized as foreign by the immune system). Sometumor cells contain specific antigens that trigger the production oftumor-specific antibodies. Large quantities of these antibodies can bemade in the laboratory and attached to radioactive substances (a processknown as radiolabeling). Once injected into the body, the antibodiesactively seek out the cancer cells, which are destroyed by thecell-killing (cytotoxic) action of the radiation. This approach canminimize the risk of radiation damage to healthy cells. The success ofthis technique will depend upon both the identification of appropriateradioactive substances and determination of the safe and effective doseof radiation that can be delivered in this way.

Radiation therapy may be used alone or in combination with chemotherapyor surgery. Like all forms of cancer treatment, radiation therapy canhave side effects. Possible side effects of treatment with radiationinclude temporary or permanent loss of hair in the area being treated,skin irritation, temporary change in skin color in the treated area, andtiredness. Other side effects are largely dependent on the area of thebody that is treated.

Hyperthermia Therapy

Hyperthermia, a procedure in which body tissue is exposed to hightemperatures (up to 106° F.), is under investigation to assess itseffectiveness in the treatment of cancer. Heat may help shrink tumors bydamaging cells or depriving them of substances they need to live.Hyperthermia therapy can be local, regional, and whole-bodyhyperthermia, using external and internal heating devices. Hyperthermiais almost always used with other forms of therapy (radiation therapy,chemotherapy, and biological therapy) to try to increase theireffectiveness.

Local hyperthermia refers to heat that is applied to a very small area,such as a tumor. The area may be heated externally with high-frequencywaves aimed at a tumor from a device outside the body. To achieveinternal heating, one of several types of sterile probes may be used,including thin, heated wires or hollow tubes filled with warm water;implanted microwave antennae; and radiofrequency electrodes.

In regional hyperthermia, an organ or a limb is heated. Magnets anddevices that produce high energy are placed over the region to beheated. In another approach, called perfusion, some of the patient'sblood is removed, heated, and then pumped (perfused) into the regionthat is to be heated internally.

Whole-body heating is used to treat metastatic cancer that has spreadthroughout the body. It can be accomplished using warm-water blankets,hot wax, inductive coils (like those in electric blankets), or thermalchambers (similar to large incubators).

Hyperthermia does not cause any marked increase in radiation sideeffects or complications. Heat applied directly to the skin, however,can cause discomfort or even significant local pain in about half thepatients treated. It can also cause blisters, which generally healrapidly. Less commonly, it can cause burns.

Photodynamic Therapy

Photodynamic therapy (also called PDT, photoradiation therapy,phototherapy, or photochemotherapy) is a treatment for some types ofcancer. It is based on the discovery that certain chemicals known asphotosensitizing agents can kill one-celled organisms when the organismsare exposed to a particular type of light. PDT destroys cancer cellsthrough the use of a fixed-frequency laser light in combination with aphotosensitizing agent.

In PDT, the photosensitizing agent is injected into the bloodstream andabsorbed by cells all over the body. The agent remains in cancer cellsfor a longer time than it does in normal cells. When the treated cancercells are exposed to laser light, the photosensitizing agent absorbs thelight and produces an active form of oxygen that destroys the treatedcancer cells. Light exposure must be timed carefully so that it occurswhen most of the photosensitizing agent has left healthy cells but isstill present in the cancer cells.

The laser light used in PDT can be directed through a fiber-optic (avery thin glass strand). The fiber-optic is placed close to the cancerto deliver the proper amount of light. The fiber-optic can be directedthrough a bronchoscope into the lungs for the treatment of lung canceror through an endoscope into the esophagus for the treatment ofesophageal cancer.

An advantage of PDT is that it causes minimal damage to healthy tissue.However, because the laser light currently in use cannot pass throughmore than about 3 centimeters of tissue (a little more than one and aneighth inch), PDT is mainly used to treat tumors on or just under theskin or on the lining of internal organs.

Photodynamic therapy makes the skin and eyes sensitive to light for 6weeks or more after treatment. Patients are advised to avoid directsunlight and bright indoor light for at least 6 weeks. If patients mustgo outdoors, they need to wear protective clothing, includingsunglasses. Other temporary side effects of PDT are related to thetreatment of specific areas and can include coughing, troubleswallowing, abdominal pain, and painful breathing or shortness ofbreath.

In December 1995, the U.S. Food and Drug Administration (FDA) approved aphotosensitizing agent called porfimer sodium, or Photofrin®, to relievesymptoms of esophageal cancer that is causing an obstruction and foresophageal cancer that cannot be satisfactorily treated with lasersalone. In January 1998, the FDA approved porfimer sodium for thetreatment of early nonsmall cell lung cancer in patients for whom theusual treatments for lung cancer are not appropriate. The NationalCancer Institute and other institutions are supporting clinical trials(research studies) to evaluate the use of photodynamic therapy forseveral types of cancer, including cancers of the bladder, brain,larynx, and oral cavity.

Laser Therapy

Laser therapy involves the use of high-intensity light to destroy cancercells. This technique is often used to relieve symptoms of cancer suchas bleeding or obstruction, especially when the cancer cannot be curedby other treatments. It may also be used to treat cancer by shrinking ordestroying tumors.

The term “laser” stands for light amplification by stimulated emissionof radiation. Ordinary light, such as that from a light bulb, has manywavelengths and spreads in all directions. Laser light, on the otherhand, has a specific wavelength and is focused in a narrow beam. Thistype of high-intensity light contains a lot of energy. Lasers are verypowerful and may be used to cut through steel or to shape diamonds.Lasers also can be used for very precise surgical work, such asrepairing a damaged retina in the eye or cutting through tissue (inplace of a scalpel).

Although there are several different kinds of lasers, only three kindshave gained wide use in medicine:

-   -   Carbon dioxide (CO2) laser—This type of laser can remove thin        layers from the skin's surface without penetrating the deeper        layers. This technique is particularly useful in treating tumors        that have not spread deep into the skin and certain precancerous        conditions. As an alternative to traditional scalpel surgery,        the CO2 laser is also able to cut the skin. The laser is used in        this way to remove skin cancers.    -   Neodymium: yttrium-aluminum-garnet (Nd:YAG) laser—Light from        this laser can penetrate deeper into tissue than light from the        other types of lasers, and it can cause blood to clot quickly.        It can be carried through optical fibers to less accessible        parts of the body. This type of laser is sometimes used to treat        throat cancers.    -   Argon laser—This laser can pass through only superficial layers        of tissue and is therefore useful in dermatology and in eye        surgery. It also is used with light-sensitive dyes to treat        tumors in a procedure known as photodynamic therapy (PDT).

Lasers have several advantages over standard surgical tools, including:

-   -   Lasers are more precise than scalpels. Tissue near an incision        is protected, since there is little contact with surrounding        skin or other tissue.    -   The heat produced by lasers sterilizes the surgery site, thus        reducing the risk of infection.    -   Less operating time may be needed because the precision of the        laser allows for a smaller incision.    -   Healing time is often shortened; since laser heat seals blood        vessels, there is less bleeding, swelling, or scarring.    -   Laser surgery may be less complicated. For example, with fiber        optics, laser light can be directed to parts of the body without        making a large incision.    -   More procedures may be done on an outpatient basis.

There are also disadvantages with laser surgery:

-   -   Relatively few surgeons are trained in laser use.    -   Laser equipment is expensive and bulky compared with the usual        surgical tools, such as scalpels.    -   Strict safety precautions must be observed in the operating        room. (For example, the surgical team and the patient must use        eye protection.)

Lasers can be used in two ways to treat cancer: by shrinking ordestroying a tumor with heat, or by activating a chemical—known as aphotosensitizing agent—that destroys cancer cells. In PDT, aphotosensitizing agent is retained in cancer cells and can be stimulatedby light to cause a reaction that kills cancer cells.

CO2 and Nd:YAG lasers are used to shrink or destroy tumors. They may beused with endoscopes, tubes that allow physicians to see into certainareas of the body, such as the bladder. The light from some lasers canbe transmitted through a flexible endoscope fitted with fiber optics.This allows physicians to see and work in parts of the body that couldnot otherwise be reached except by surgery and therefore allows veryprecise aiming of the laser beam. Lasers also may be used with low-powermicroscopes, giving the doctor a clear view of the site being treated.Used with other instruments, laser systems can produce a cutting area assmall as 200 microns in diameter—less than the width of a very finethread.

Lasers are used to treat many types of cancer. Laser surgery is astandard treatment for certain stages of glottis (vocal cord), cervical,skin, lung, vaginal, vulvar, and penile cancers.

In addition to its use to destroy the cancer, laser surgery is also usedto help relieve symptoms caused by cancer (palliative care). Forexample, lasers may be used to shrink or destroy a tumor that isblocking a patient's trachea (windpipe), making it easier to breathe. Itis also sometimes used for palliation in colorectal and anal cancer.

Laser-induced interstitial thermotherapy (LITT) is one of the mostrecent developments in laser therapy. LITT uses the same idea as acancer treatment called hyperthermia; that heat may help shrink tumorsby damaging cells or depriving them of substances they need to live. Inthis treatment, lasers are directed to interstitial areas (areas betweenorgans) in the body. The laser light then raises the temperature of thetumor, which damages or destroys cancer cells.

Gene Therapy

Gene therapy is an experimental medical intervention that involvesmodifying the genetic material of living cells to fight disease. Genetherapy is being studied in clinical trials (research studies withhumans) for many different types of cancer and for other diseases.

One of the goals of gene therapy is to supply cells with healthy copiesof missing or altered genes. Instead of giving a patient a drug, doctorsattempt to correct the problem by altering the genetic makeup of some ofthe patient's cells. Examples of diseases that could be treated this wayinclude cystic fibrosis and hemophilia.

Gene therapy is also being studied as a way to change how a cellfunctions; for example, by stimulating immune system cells to attackcancer cells.

In general, a gene is delivered to the cell using a “vector.” The mostcommon types of vectors used in gene therapy are viruses. Viruses usedas vectors in gene therapy are genetically disabled; they are unable toreproduce themselves. Most gene therapy clinical trials rely on mouseretroviruses to deliver the desired gene. Other viruses used as vectorsinclude adenoviruses, adeno-associated viruses, poxviruses, and theherpes virus.

A gene therapy can be done both ex vivo and in vivo. In most ex vivogene therapy clinical trials, cells from the patient's blood or bonemarrow are removed and grown in the laboratory. The cells are exposed tothe virus that is carrying the desired gene. The virus enters the cells,and the desired gene becomes part of the cells' DNA. The cells grow inthe laboratory and are then returned to the patient by injection into avein. In in vivo gene therapy, vectors or liposomes are used to deliverthe desired gene to cells inside the patient's body.

Immunotherapy

Cancer may develop when the immune system breaks down or is notfunctioning adequately. Immunotherapy uses the body's immune system,either directly or indirectly, to fight cancer or to lessen the sideeffects that may be caused by some cancer treatments. Immunotherapy isdesigned to repair, stimulate, or enhance the immune system's responses.

Immune system cells include the following: Lymphocytes are a type ofwhite blood cell found in the blood and many other parts of the body.Types of lymphocytes include B cells, T cells, and Natural Killer cells.B cells (B lymphocytes) mature into plasma cells that secrete antibodies(immunoglobulins), the proteins that recognize and attach to foreignsubstances known as antigens. Each type of B cell makes one specificantibody, which recognizes one specific antigen. T cells (T lymphocytes)directly attack infected, foreign, or cancerous cells. T cells alsoregulate the immune response by signaling other immune system defenders.T cells work primarily by producing proteins called lymphokines. NaturalKiller cells (NK cells) produce powerful chemical substances that bindto and kill any foreign invader. They attack without first having torecognize a specific antigen. Monocytes are white blood cells that canswallow and digest microscopic organisms and particles in a processknown as phagocytosis. Monocytes can also travel into tissue and becomemacrophages.

Cells in the immune system secrete two types of proteins: antibodies andcytokines. Antibodies respond to antigens by latching on to, or bindingwith, the antigens. Specific antibodies match specific antigens, fittingtogether much the way a key fits a lock. Cytokines are substancesproduced by some immune system cells to communicate with other cells.Types of cytokines include lymphokines, interferons, interleukins, andcolony-stimulating factors. Cytotoxic cytokines are released by a typeof T cell called a cytotoxic T cell. These cytokines attack cancer cellsdirectly.

Nonspecific immunomodulating agents are substances that stimulate orindirectly augment the immune system. Often, these agents target keyimmune system cells and cause secondary responses such as increasedproduction of cytokines and immunoglobulins. Two nonspecificimmunomodulating agents used in cancer treatment are bacillusCalmette-Guerin (BCG) and levamisole. BCG, which has been widely used asa tuberculosis vaccine, is used in the treatment of superficial bladdercancer following surgery. BCG may work by stimulating an inflammatory,and possibly an immune, response. A solution of BCG is instilled in thebladder and stays there for about 2 hours before the patient is allowedto empty the bladder by urinating. This treatment is usually performedonce a week for 6 weeks. Levamisole is used along with fluorouracil(5-FU) chemotherapy in the treatment of stage III (Dukes' C) coloncancer following surgery. Levamisole may act to restore depressed immunefunction.

Some antibodies, cytokines, and other immune system substances can beproduced in the laboratory for use in cancer treatment. These substancesare often called biological response modifiers (BRMs). They alter theinteraction between the body's immune defenses and cancer cells toboost, direct, or restore the body's ability to fight the disease. BRMsinclude interferons, interleukins, colony-stimulating factors,monoclonal antibodies, and vaccines. Immunotherapy may be used to stop,control, or suppress processes that permit cancer growth; make cancercells more recognizable, and therefore more susceptible, to destructionby the immune system; boost the killing power of immune system cells,such as T cells, NK cells, and macrophages; alter cancer cells' growthpatterns to promote behavior like that of healthy cells; block orreverse the process that changes a normal cell or a precancerous cellinto a cancerous cell; enhance the body's ability to repair or replacenormal cells damaged or destroyed by other forms of cancer treatment,such as chemotherapy or radiation; and prevent cancer cells fromspreading to other parts of the body.

Some BRMs are a standard part of treatment for certain types of cancer,while others are being studied in clinical trials. BRMs are being usedalone or in combination with each other. They are also being used withother treatments, such as radiation therapy and chemotherapy.

Interferons (IFNs) are types of cytokines that occur naturally in thebody. They were the first cytokines produced in the laboratory for useas BRMs. There are three major types of interferons—interferon alpha,interferon beta, and interferon gamma; interferon alpha is the type mostwidely used in cancer treatment. Interferons can improve the way acancer patient's immune system acts against cancer cells. In addition,interferons may act directly on cancer cells by slowing their growth orpromoting their development into cells with more normal behavior. Someinterferons may also stimulate NK cells, T cells, and macrophages,boosting the immune system's anticancer function. The U.S. Food and DrugAdministration (FDA) has approved the use of interferon alpha for thetreatment of certain types of cancer, including hairy cell leukemia,melanoma, chronic myeloid leukemia, and AIDS-related Kaposi's sarcoma.Studies have shown that interferon alpha may also be effective intreating other cancers such as metastatic kidney cancer andnon-Hodgkin's lymphoma.

Like interferons, interleukins (IL) are cytokines that occur naturallyin the body and can be made in the laboratory. Many interleukins havebeen identified; interleukin-2 (IL-2 or aldesleukin) has been the mostwidely studied in cancer treatment. IL-2 stimulates the growth andactivity of many immune cells, such as lymphocytes, that can destroycancer cells. The FDA has approved IL-2 for the treatment of metastatickidney cancer and metastatic melanoma.

Colony-stimulating factors (CSFs) (sometimes called hematopoietic growthfactors) usually do not directly affect tumor cells; rather, theyencourage bone marrow stem cells to divide and develop into white bloodcells, platelets, and red blood cells. Bone marrow is critical to thebody's immune system because it is the source of all blood cells. TheCSFs' stimulation of the immune system may benefit patients undergoingcancer treatment. Because anticancer drugs can damage the body's abilityto make white blood cells, red blood cells, and platelets, patientsreceiving anticancer drugs have an increased risk of developinginfections, becoming anemic, and bleeding more easily. By using CSFs tostimulate blood cell production, doctors can increase the doses ofanticancer drugs without increasing the risk of infection or the needfor transfusion with blood products. CSFs are particularly useful whencombined with high-dose chemotherapy. Some examples of CSFs and theiruse in cancer therapy are as follows: G-CSF (filgrastim) and GM-CSF(sargramostim) can increase the number of white blood cells, therebyreducing the risk of infection in patients receiving chemotherapy. G-CSFand GM-CSF can also stimulate the production of stem cells inpreparation for stem cell or bone marrow transplants; Erythropoietin canincrease the number of red blood cells and reduce the need for red bloodcell transfusions in patients receiving chemotherapy; and Oprelvekin canreduce the need for platelet transfusions in patients receivingchemotherapy.

CSFs are used in clinical trials to treat some types of leukemia,metastatic colorectal cancer, melanoma, lung cancer, and other types ofcancer.

Monoclonal Antibodies (MOABs) are also being evaluated in cancertherapy. These antibodies are produced by a single type of cell and arespecific for a particular antigen. MOABs specific to the antigens foundon the surface of the cancer cell being treated are being created.

MOABs are made by injecting human cancer cells into mice so that theirimmune systems will make antibodies against these cancer cells. Themouse cells producing the antibodies are then removed and fused withlaboratory-grown cells to create “hybrid” cells called hybridomas.Hybridomas can indefinitely produce large quantities of these pureantibodies, or MOABs. MOABs may be used in cancer treatment in a numberof ways: MOABs that react with specific types of cancer may enhance apatient's immune response to the cancer. MOABs can be programmed to actagainst cell growth factors, thus interfering with the growth of cancercells. MOABs may be linked to anticancer drugs, radioisotopes(radioactive substances), other BRMs, or other toxins. When theantibodies latch onto cancer cells, they deliver these poisons directlyto the tumor, helping to destroy it. MOABs may help destroy cancer cellsin bone marrow that has been removed from a patient in preparation for abone marrow transplant. MOABs carrying radioisotopes may also proveuseful in diagnosing certain cancers, such as colorectal, ovarian, andprostate.

Rituxan® (rituximab) and Herceptin® (trastuzumab) are examples ofmonoclonal antibodies that have been approved by the FDA. Rituxan isused for the treatment of B-cell non-Hodgkin's lymphoma that hasreturned after a period of improvement or has not responded tochemotherapy. Herceptin is used to treat metastatic breast cancer inpatients with tumors that produce excess amounts of a protein calledHER-2. (Approximately 25 percent of breast cancer tumors produce excessamounts of HER-2.) MOABs are begun tested in clinical trials to treatlymphomas, leukemias, colorectal cancer, lung cancer, brain tumors,prostate cancer, and other types of cancer.

Cancer vaccines are another form of immunotherapy currently under study.Vaccines for infectious diseases, such as measles, mumps, and tetanus,are effective because they expose the body's immune cells to weakenedforms of antigens that are present on the surface of the infectiousagent. This exposure causes the immune cells to produce more plasmacells, which make antibodies. T cells that recognize the infectiousagent also multiply. These activated T cells later remember theexposure. The next time the agent enters the body, cells in the immunesystem are already prepared to respond and stop the infection.

For cancer treatment, researchers are developing vaccines that mayencourage the patient's immune system to recognize cancer cells. Thesevaccines may help the body reject tumors and prevent cancer fromrecurring. In contrast to vaccines against infectious diseases, cancervaccines are designed to be injected after the disease is diagnosed,rather than before it develops. Cancer vaccines given when the tumor issmall may be able to eradicate the cancer. Early cancer vaccine clinicaltrials (research studies with people) involved mainly patients withmelanoma. Currently, cancer vaccines are also being studied in thetreatment of many other types of cancer, including lymphomas and cancersof the kidney, breast, ovary, prostate, colon, and rectum. Researchersare also investigating ways that cancer vaccines can be used incombination with other BRMs.

Like other forms of cancer treatment, biological therapies can cause anumber of side effects, which can vary widely from patient to patient.Rashes or swelling may develop at the site where the BRMs are injected.Several BRMs, including interferons and interleukins, may cause flu-likesymptoms including fever, chills, nausea, vomiting, and appetite loss.Fatigue is another common side effect of BRMs. Blood pressure may alsobe affected. The side effects of IL-2 can often be severe, depending onthe dosage given. Patients need to be closely monitored duringtreatment. Side effects of CSFs may include bone pain, fatigue, fever,and appetite loss. The side effects of MOABs vary, and serious allergicreactions may occur. Cancer vaccines can cause muscle aches and fever.

Sensitizing Compositions and Methods—Dosage, Mode of Administration, andPharmaceutical Formulations

The present invention provides for a composition comprising a SPARCfamily polypeptide or a polynucleotide encoding such polypeptide and atherapeutic agent. The SPARC polypeptide or polynucleotide is providedas a therapeutically effective amount so as to sensitize a cancer cellor patient to the treatment by said therapeutic agent.

The therapeutic agent may be any suitable agent for a specific therapyas described herein and as known in the art. It may be a chemotherapyagent, i.e., a drug, for example, 5-fluorouracil; it may be a radiationagent, such as a radiolabeled antibody, a radiosensitizer, or aradioactive seed implant. The therapeutic agent may also be aphotosensitizing agent, such as porfimer sodium; or a gene therapy agent(e.g., a vector), or it may be an immunotherapy agent, such as a immunecell, an antibody, or cytokine.

The present invention provides a composition comprising a SPARC familypolypeptide and a chemotherapy-resistant cell.

The present invention also provides a recombinant cell comprising aheterologous transcription control region operatively associated with aSPARC family polynucleotide.

In addition to sensitizing a sample or a mammal to cancer therapy, theuse of the subject compositions of the present invention can reduce thedosage of a therapy, therefore reducing the side effects caused bycancer therapy.

The above compositions may be a pharmaceutical composition whichincludes a pharmaceutically acceptable carrier or excipient.

As used herein, a “carrier” refers to any substance suitable as avehicle for delivering an APC to a suitable in vitro or in vivo site ofaction. As such, carriers can act as an excipient for formulation of atherapeutic or experimental reagent containing an APC. Preferredcarriers are capable of maintaining an APC in a form that is capable ofinteracting with a T cell. Examples of such carriers include, but arenot limited to water, phosphate buffered saline, saline, Ringer'ssolution, dextrose solution, serum-containing solutions, Hank's solutionand other aqueous physiologically balanced solutions or cell culturemedium. Aqueous carriers can also contain suitable auxiliary substancesrequired to approximate the physiological conditions of the recipient,for example, enhancement of chemical stability and isotonicity. Suitableauxiliary substances include, for example, sodium acetate, sodiumchloride, sodium lactate, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, and other substances used toproduce phosphate buffer, Tris buffer, and bicarbonate buffer.

A composition comprising a SPARC family polypeptide and a therapeuticagent may be used to sensitize a cancer in vitro by directly contactingthe cancer sample with an effective amount of the purified SPARC familypolypeptide. A mammal (e.g., a cancer patient) can be administered acomposition comprising a SPARC family polypeptide to achieve thesensitizing effect in vivo. In addition, a cancer sample, either cellsor tissue, may be obtained from the mammal and sensitized using theSPARC family polypeptide ex vivo before being returned back to themammal.

A SPARC family polynucleotide of the present invention may be introducedinto a cancer cell in vitro to sensitizing the response of the cancercell, or it may be delivered to a mammal in vivo through an appropriatevector as known in the art and as described herein above. In addition,the polynucleotide may be introduced ex vivo into cancer cells or tissueobtained from a mammal in need, and the cells or tissue then returned tothe mammal in need. Being a secreted protein, a SPARC family polypeptidemade by such ex vivo introduced cells may function in the localenvironment to sensitize not only the modified cells, but also theneighboring non-modified cancer cells.

A composition comprising a recombinant cell may be introduced into amammal for sensitizing treatment.

Subject dose size, number of doses, frequency of dose administration,and mode of administration can be determined and optimized using methodsknown in the art (see, e.g., Hardman et al., Ceds 1995, Goodman &Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition,McGraw-Hill).

Dosages of each therapy in treating various cancer patients are known inthe art and can be determined by a skilled physician. For example, asuitable SPARC polypeptide dose may be in the range of 0.01 to 100 mgSPARC polypeptide per kilogram body weight of the recipient per day,preferably in the range of 0.2 to 10 mg per kilogram body weight perday. A SPARC polynucleotide of the present invention may be administeredat a suitable dose in the range of 0.01 to 100 mg polynucleotide perkilogram body weight of the recipient per day, preferably in the rangeof 0.2 to 10 mg per kilogram body weight per day. The cells comprising arecombinant SPARC polynucleotide may be administered at a dosage in therange of 104-1010 per kilogram body weight of the recipient, preferablyin the range of 106-108 per kilogram body weight of the recipient. Thedesired dose is preferably presented once daily, but may be dosed astwo, three, four, five, six or more sub-doses administered atappropriate intervals throughout the day. These sub-doses may beadministered in unit dosage forms, for example, containing 10 to 1500mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of theSPARC family polypeptide per unit dosage form. Dosages of the SPARCfamily polypeptide or the SPARC family polynucleotide, or the cellscomprising a recombinant SPARC family polynucleotide useful according tothe invention will vary depending upon the condition to be treated orprevented and on the identity of the SPARC family polypeptide orpolynucleotide being used. Estimates of effective dosages and in vivohalf-lives for the individual composition encompassed by the inventioncan be made on the basis of in vivo testing using an animal model, suchas the mouse model described herein or an adaptation of such method tolarger mammals.

In Vitro/Ex Vivo Applications

Compositions provided by the present invention may be used to sensitizea cancer cell in vitro using methods known in the art, and as describedherein before. Thus the present invention provides a method forsensitizing a cancer cell to a therapeutic treatment, the methodcomprising contacting the cancer sample with an effective amount of acomposition comprising a SPARC family polypeptide or a polynucleotideencoding a SPARC family polypeptide. The present invention also providesa method for ex vivo sensitizing a mammal diagnosed with cancer to atherapeutic treatment, the method comprising: (1) Obtaining a cancersample from the mammal; (2) contacting the cancer sample with aneffective amount of a composition comprising a SPARC family polypeptideor a polynucleotide encoding a SPARC family polypeptide; and (3)returning the cancer sample after the contacting of (2) to the mammal.

Ex vivo gene therapy refers to the isolation of cells from an animal,the delivery of a polynucleotide into the cells, in vitro, and then thereturn of the modified cells back into the animal. This may involve thesurgical removal of tissue/organs from an animal or the primary cultureof cells and tissues. Anderson et al., U.S. Pat. No. 5,399,346, andincorporated herein in its entirety, disclose ex vivo therapeuticmethods. This method is applicable because a SPARC family polypeptide isa secreted polypeptide. The return of the modified cells back to amammal may increase the extracellular concentration of a SPARC familypolypeptide locally and therefore sensitizing the unmodified cancercells which are in proximity with the modified cells.

When tissue sample needs to be taken from a mammal for ex vivoapplication, cellular extracts may be prepared from tissue biopsies ofpatients including, but not limited to brain, heart, lung, lymph nodes,eyes, joints, skin and neoplasms associated with these organs. “Tissuebiopsy” also encompasses the collection of biological fluids includingbut not limited to blood, plasma, sputum, urine, cerebrospinal fluid,lavages, and leukophoresis samples. In a preferred embodiment, “tissuebiopsies” according to the invention are taken from tumors of thebreast, ovary or prostate. “Tissue biopsies” are obtained usingtechniques well known in the art including needle aspiration and punchbiopsy of the skin.

Generally, when a polynucleotide is introduced into cells in culture(e.g., by one of the transfection techniques described above) only asmall fraction of cells (about 1 out of 10⁵) typically integrate thetransfected polynucleotide into their genomes (i.e., the polynucleotideis maintained in the cell episomally). Thus, in order to identify cellswhich have taken up exogenous polynucleotide, it is advantageous totransfect polynucleotide encoding a selectable marker into the cellalong with the polynucleotide(s) of interest, i.e., a SPARC familypolynucleotide, as described herein before.

In Vivo Applications

The composition provided by the present invention can be administered toa mammal, e.g., in a method of sensitizing a therapeutic treatment. Thusthe present invention provides a method for in vivo sensitizing a mammaldiagnosed with cancer to a therapeutic treatment, the method comprisingadministrating to the mammal an effective amount of a compositioncomprising a SPARC family polypeptide or a polynucleotide encoding aSPARC family polypeptide.

The manner of administration of a composition of the present inventioncan depend upon the particular purpose for the delivery, the overallhealth and condition of the patient and the judgment of the physician ortechnician administering the target vehicle. A composition of thepresent invention can be administered to an animal using a variety ofmethods. Such delivery methods can include parenteral, topical, oral orlocal administration, such as intradermally. A composition can beadministered in a variety of unit dosage forms depending upon the methodof administration. Preferred delivery methods for a composition of thepresent invention include intravenous administration, localadministration (e.g., intra-tumoral) by, for example, injection,intradermal injection, intramuscular injection, intraperitonealinjection and inhalation. For particular modes of delivery, acomposition of the present invention can be formulated in an excipientof the present invention. A composition of the present invention can beadministered to any animal, preferably to mammals, and more preferablyto humans.

Injection: The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral administration: Pharmaceutical compositions for oral administrationare formulated using pharmaceutically acceptable carriers well known inthe art in dosages suitable for oral administration. Such carriersenable the pharmaceutical compositions to be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for ingestion by the patient.

Pharmaceutical preparations for oral use are obtained through acombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which are used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Nasal administration: For nasal administration, penetrants appropriateto the particular barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Subcutaneous and intravenous use: For subcutaneous and intravenous use,the composition of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity. Suitable aqueous vehicles include Ringer's solution andisotonic sodium chloride. Aqueous suspensions according to the inventionmay include suspending agents such as cellulose derivatives, sodiumalginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agentsuch as lecithin. Suitable preservatives for aqueous suspensions includeethyl and n-propyl p-hydroxybenzoate.

The composition useful according to the invention may also be presentedas liposome formulations.

Gene therapy using the compositions provided by the present inventionmay be carried out according to generally accepted methods, for example,as described by Friedman in “Therapy for Genetic Disease,” T. Friedman,ed., Oxford University Press (1991), pp. 105-121, hereby incorporated byreference.

The pharmaceutical compositions of the present invention may bemanufactured in a manner known in the art, e.g. by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

After pharmaceutical compositions comprising a therapeutic agent of theinvention formulated in a acceptable carrier have been prepared, theyare placed in an appropriate container and labeled for treatment of anindicated condition with information including amount, frequency andmethod of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state (e.g., location of the disease, age,weight, and gender of the patient, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy). Long acting pharmaceutical compositionsmight be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularformulation. General guidance as to particular dosages and methods ofdelivery for other applications is provided in the literature (see U.S.Pat. Nos. 4,657,760; 5,206,344; and 5,225,212, herein incorporated byreference). Those skilled in the art will typically employ differentformulations for oligonucleotides and gene therapy vectors than forproteins or their inhibitors. Similarly, delivery of polynucleotides orpolypeptides will be specific to particular cells, conditions,locations, and the like.

The composition provided by the present invention may be formulated asdescribed in U.S. Pat. No. 6,187,330 (hereby incorporated by referencein its entirety) which provides a composition for the controlled releaseof a peptide or protein comprising a biocompatible, bioerodable polymerhaving dispersed therein a glassy matrix phase comprising the peptide orprotein and a thermoprotectant, said glassy matrix phase having a glasstransition temperature above the melting point of the polymer. Since thepeptide or protein drug is stable within the composition, it canconveniently be formed, in its melt stage, into suitably shaped devicesto be used as drug delivery implants, e.g. in the form of rods, films,beads or other desired shapes.

Determining Resistance or Sensitivity to a Therapeutic Treatment

The determination of a cancer sample (e.g., cells or tissue) respondingto a therapeutic treatment can be carried out by any methods known inthe art. For example, by a cell culture drug resistance testing (CCDRT).CCDRT refers to testing a cancer sample (e.g., taken from a mammalpatient) in the laboratory to drugs that may be used to treat thepatient's cancer. The testing can identify the cancer sample issensitive to which drugs and resistant to which drugs, which indicateswhich drugs are more likely to work and which drugs are less likely towork in the patient. The sensitivity of the cancer sample (therefore,the patient) can be sensitized by treatment with the compositionprovided by the present invention. CCDRT can be performed again with thesensitizing treatment and determine if a sensitizing compositionprovided by the present invention can sensitize the response of thecancer sample to a specific treatment or not. A composition can be saidto sensitize a therapeutic treatment if the response as measured byCCDRT is increased by at least 20%, e.g., 30%, 40%, 50% 80%, 100%(2-fold), or 3-fold, 4-fold, 5-fold, or more when compared to theresponse in the absence of the sensitizing composition.

CCDRT typically include the cell proliferation assays and cell deathassays.

The cell proliferation assay measures the proliferation of cells. It canbe done by the radioactive thymidine incorporation assay originallydescribed by Tanigawa and Kern (supra). In this assay, applied only tosolid tumors and not to hematologic neoplasms, tumor cells suspended insoft agarose are cultured for 4-6 days in the continuous presence ofantineoplastic drugs. At the end of the culture period, radioactivethymidine is introduced and differences in putative thymidineincorporation into DNA are compared between control and drug-treatedcultures. Kern and Weisenthal analyzed the clinical correlation data anddefined the concept of “extreme drug resistance,” or EDR [Kern D H,Weisenthal L M. J Natl Cancer Inst 1990; 82: 582-588]. This was definedas an assay result which was one standard deviation more resistant thanthe median result for comparison, database assays. Patients treated withsingle agents showing EDR in the assay virtually never enjoyed a partialor complete response. Kern and Weisenthal also defined “low drugresistance” (LDR) as a result less resistant than the median and“intermediate drug resistance” (IDR) as a result more resistant than themedian but less resistant than EDR (in other words, between the medianand one standard deviation more resistant than the median).

The principles and clinical correlation data with the thymidine “EDR”assay were reviewed in 1992 (Weisenthal L M, Kern D H. Oncology (USA)1992; 5: 93-103]. There have been only a few follow-up studies publishedsince this time. One such study showed that EDR to one or more of thesingle agents used in a two drug combination is not apparentlyassociated with a lower probability of response to the two drugcombination in the setting of intraperitoneal chemotherapy ofappendiceal and colon cancers (Fernandez-Trigo V, Shamsa F, Vidal-JoveJ, Kern D H, Sugarbaker P H. Am J Clin Oncol 1995; 18: 454-460). It is,however, possible that response to the high drug concentrationsachievable with intraperitoneal chemotherapy may be more closelyassociated with drug penetration to the tumor than to intrinsic drugresistance of the tumor cells. It was also shown that EDR to paclitaxeldoes not appear to be a prognostic factor in ovarian cancer patients orin patients with primary peritoneal carcinoma treated with paclitaxelplus platinum (Eltabbakh G H, Piver M S, Hempling R E, et al. GynecolOncol 1998; 70: 392-397; Eltabbakh G H. J Surg Oncol 2000; 73: 148-152).However, it was recently reported that EDR to platinum in ovarian cancermay have prognostic implications (Fruehauf, J., et al. Proc ASCO, v. 20,Abs 2529, 2001). It was also reported that previously-untreated breastcancer patients with tumors showing LDR (defined above) had superiortimes to progression and overall survivals than patients with tumorsshowing either IDR or EDR (Mehta, R. S., et al, Breast Cancer Res Treat66:225-37, 2001).

The thymidine “EDR” assay has a very high specificity (>98%) for theidentification of inactive single agents, but a low sensitivity (<40%).In other words, a drug with assay-defined “EDR” is predicted to bealmost certain to be inactive as a single agent (high specificity foridentifying inactive drugs), but many drugs without “EDR” will also beinactive (low sensitivity for identifying inactive drugs).

A second form of cell proliferation assay is the adhesive tumor cellculture system, based on comparing monolayer growth of cells over aproprietary “cell adhesive matrix” (Ajani J A, Baker F L, Spitzer G, etal. J Clin Oncol 1987). Positive clinical correlations were alsodescribed in this publication.

In some embodiments, colony formation assays are used to measure cellproliferation. In this test cells are grown in vitro in soft agar(tissue culture medium containing agar as a gelling agent; also referredto as semi-solid agar) or other highly viscous media, containing, forexample, methylcellulose, plasma gel or fibrin clots. These semi-solidmedia reduce cell movement and allow individual cells to develop intocell clones that are identified as single colonies. These assays arealso generally referred to as Clonogenic assays. The colony formationassays are well known in the art, for example, see Rizzino, A Soft agargrowth assays for transforming growth factors and mitogenic peptides.Methods in Enzymology 146: 341-53 (1987) and In some embodiments,apoptosis is measured by a terminal deoxynucleotide transferase-mediateddUTP nick end labeling (TUNEL) assay which is well known in the art andMaterials and Methods available as supporting online material on ScienceOnline.

As opposed to measuring cell proliferation, there is a closely-relatedfamily of assays based on the concept of total cell kill, or, in otherwords, cell death occurring in the population of tumor cells (WeisenthalL M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M. RecentResults Cancer Res 1984; 94: 161-173; Weisenthal L M, Lippman M E.Cancer Treat Rep 1985; 69: 615-632; Weisenthal L M. Cell culture assaysfor hematologic neoplasms based on the concept of total tumor cell kill.In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A JP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, Pa.:Harwood Academic Publishers, 1993: 415-432; Weisenthal L M. ContribGynecol Obstet 1994; 19: 82-90). The concepts underlying cell deathassays are relatively simple, even though the technical features anddata interpretation can be very complex.

The basic technology concepts are straightforward. For example, a freshspecimen is obtained from a viable neoplasm. The specimen is most oftena surgical specimen from a viable solid tumor. Less often, it is amalignant effusion, bone marrow, or peripheral blood specimen containing“tumor” cells (a word used to describe cells from either a solid orhematologic neoplasm). These cells are isolated and then cultured in thecontinuous presence or absence of drugs, most often for 3 to 7 days. Atthe end of the culture period, a measurement is made of cell injury,which correlates directly with cell death. There is evidence that themajority of available anticancer drugs may work through a mechanism ofcausing sufficient damage to trigger so-called programmed cell death, orapoptosis (Hickman J A. Cancer Metastasis Rev 1992; 11: 121-139; ZuninoF, Perego P, Pilotti S, Pratesi G, Supino R, Arcamone F. Pharmacol Ther1997; 76: 177-185).

Although there are methods for specifically measuring apoptosis whichcan be applied to cultured cells, there are practical difficulties inapplying these methods to mixed (and clumpy) populations of tumor cellsand normal cells. Thus, more general measurements of cell death havebeen applied. These include: (1) delayed loss of cell membrane integrity(which has been found to be a useful surrogate for apoptosis), asmeasured by differential staining in the DISC assay method, which allowsselective drug effects against tumor cells to be recognized in a mixedpopulation of tumor and normal cells (Weisenthal L M, Kern D H. Oncology(USA) 1992; 5: 93-103; Weisenthal L M, Marsden J A, Dill P L, Macaluso CK. Cancer Res 1983; 43: 749-757), (2) loss of mitochondrial Krebs cycleactivity, as measured in the MTT assay (Carmichael J, DeGraff W G,Gazdar A F, Minna J D, Mitchell J B. Cancer Res 1987; 47: 936-942), (3)loss of cellular ATP, as measured in the ATP assay (Kangas L, GronroosM, Nieminen A L. Med Biol 1984; 62: 338-343; Garewal H S, Ahmann F R,Schifman R B, Celniker A. J Natl Cancer Inst 1986; 77: 1039-1045; SevinB-U, Peng Z L, Perras J P, Ganjei P, Penalver M, Averette H E. GynecolOncol 1988; 31: 191-204), and (4) loss of cell membrane esteraseactivity and cell membrane integrity, as measured by the fluoresceindiacetate assay (Rotman B, Teplitz C, Dickinson K, Cozzolino J P. Invitro Cell Dev Biol 1988; 24: 1137-1138; Larsson R, Nygren P, Ekberg M,Slater L. Leukemia 1990; 4: 567-571; Nygren P, Kristensen J, Jonsson B,et al. Leukemia 1992; 6: 1121-1128).

In some embodiments, apoptosis is measured by a terminal deoxynucleotidetransferase-mediated dUTP nick end labeling (TUNEL) assay which is wellknown in the art and for example as described in Materials and Methodsavailable as supporting online material on Science Online.

In addition, the sensitivity or resistance of an animal to a treatmentmay be directly determined by measuring tumor size before and aftertreatment and/or over a period of time of treatment. If tumor size isdecreased by 50%, preferably 75%, more preferably 85%, most preferably100% with a treatment, than the animal is said to be sensitive (notresistant) to the treatment. Otherwise, the animal is considered to beresistant to the treatment. If the tumor size is reduced by at least25%, preferably 50%, more preferably 75%, most preferably 100% after theadministering of a treatment sensitizing composition of the presentinvention compared to the tumor after treatment but in the absence ofthe a composition of the present invention, then the composition is saidto be effective in sensitizing the treatment in the animal. In human,the tumor size may be compared over a window of 6 month period oftreatment, in other animals, this window varies for example a 4-6 weekwindow may be used for mouse. It is understood that the actual timewindow for comparing tumor size may be determined according to knowledgein the art and the particular tumor to be treated.

Furthermore, whether a cell is resistant to a treatment may be alsodetermined by measuring the expression of a SPARC family polypeptide orpolynucleotide as described herein before. Thus the present inventionprovides a method for evaluating a first cancer sample for itsresistance to a therapeutic treatment, comprising: (a) measuringexpression level of a SPARC family mRNA or polypeptide, or extracellularlevel of a SPARC family polypeptide in the first cancer sample; (b)measuring expression level of the SPARC family mRNA or polypeptide, orextracellular level of the SPARC family polypeptide in a second cancersample which does not exhibit resistance to the therapeutic treatment;(c) comparing the expression levels or the extracellular levels obtainedin (a) and (b), wherein a lower level of expression or extracellularlevel in (a) is indicative of the first cancer sample being resistant tothe therapeutic treatment.

The present invention further provides a method for identifying an agentwhich modulates a SPARC family mRNA or polypeptide expression, or aSPARC family polypeptide secretion, comprising: (a) measuring expressionlevel of the SPARC family mRNA or polypeptide, or extracellular level ofthe SPARC family polypeptide in a sample; (b) contacting a candidateagent with the sample; (c) after the contacting of (b), measuringexpression or extracellular level of the SPARC family mRNA orpolypeptide, or extracellular level of the SPARC family polypeptide inthe sample of (b); (d) comparing the expression levels or theextracellular levels in (a) and (c), wherein a differential level ofexpression or extracellular level in (a) and (c) indicates the candidateagent being an agent which modulates the SPARC family mRNA orpolypeptide expression, or the SPARC family polypeptide secretion.

Expression levels of a SPARC polypeptide or a polynucleotide and thesecretion levels of a SPARC polypeptide can be measured as describedherein before and by any method known in the art.

An agent, which enhances the expression or secretion of a SPARC familymember, may itself be used as a therapy sensitizing agent as describedin the present invention. The agent may be a chemical, or a biologicalmolecule (e.g., a protein, or a polynucleotide, etc.)

Animal Models

The therapeutic effects of the compositions provided by the presentinvention may be tested in various animal models. This may be done invitro, ex vivo, or in vivo as described herein before.

Mouse models for proliferative disorders are known in the art and can befound, for example, on Jackson laboratory mouse database at world wideweb www.jax.org and The Jackson Laboratory catalog—Jax-Mice—June2001-May 2003, or Jackson-Grusby L. 2002, Oncogene. 12; 21(35):5504-14;Ghebranious N, Donehower L A., 1998, Oncogene. 24; 17(25):3385-400;Palapattu G S, Bao S, Kumar T R, Matzuk M M. 1998, Cancer Detect Prey.22(1):75-86). For example, tumor mouse models include those used for thestudy of Chronic Myelogenous Leukemia (CML), defects in cell cdhesionmolecules, genes regulating growth and proliferation, growthfactors/receptors/cytokines, increased tumor incidence, oncogenes,toxicology and tumor suppressor genes.

EXAMPLES

The invention is based on the observation that SPARC was found to besignificantly underexpressed in chemotherapy resistant cells, that SPARCpolypeptide sensitizes cancer therapy resistant cells to cancertreatment, that SPARC encoding DNA sensitizes cells to cancer therapy,and that animals engrafted with SPARC transfectant cells show a dramaticreduction in tumor growth compared to animals engrafted with a control.

Example 1 Materials and Methods

Cell Culture—The colorectal cell line MIP-101 was maintained inDulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin/streptomycin(Invitrogen) at 37° C. and 5% CO2. Resistant MIP101 cells were developedfollowing long-term incubation with incremental concentrations of5-fluorouracil (5-FU), irinotecan (CPT-11), cisplatin (CIS), andetoposide (ETO). Stable MIP101 cells transduced with SPARC (MIP/SP) weremaintained in DMEM supplemented with 10% FBS, 1%penicillin/streptomycin, and 0.1% Zeocin at 37° C. and 5% CO₂.

Analytical Reverse Transcription-Polymerase Chain Reaction—Total RNA wasextracted from cultured cells (2×10⁶ cells, 75% confluence) using TRIZOLreagent (Invitrogen) according to the manufacturer's protocol. RT-PCRwas performed using a commercially available kit (BD Biosciences)following the manufacturer's protocol using 1 ug of total RNA. Thespecific primers used to amplify SPARC: 5′CGA AGA GGA GGT GGT GGC GGAAA-3′ (sense) (SEQ ID NO. 78) and 5′GGT TGT TGT CCT CAT CCC TCT CATAC-3′ (antisense) (SEQ ID NO. 79). GAPDH: 5′-CTC TCT GCT CCT CCT GTT CGACAG-3′ (sense) (SEQ ID NO. 80) and 5′-AGG GGT CTT ACT CCT TGG AGG CCA-3′(antisense) (SEQ ID NO. 81) was used as internal control and tonormalize the gene expression levels. The following settings were usedfor the reaction: 50° C.×1 hr, followed by 37 cycles of 94° C.×1 min,65° C.×1 min, 72° C.×2 min, followed by 72° C.×10 min and incubation at4° C. The PCR products were separated on 1% agarose gel in TAE buffer(stained with ethidium bromide 0.5 ug/ml) by electrophoresis for 1 hr at100 V and subsequently photographed.

Quantitation of Apoptosis—For the TUNEL assay, cells were plated ontoglass coverslips in 6-well plates at 250,000 cells/plate overnight priorto any induction study 24 hours later. For the assessment of apoptosisfollowing exogenous SPARC (Haematologic Technologies Inc), cells wereincubated with SPARC 5 μg/ml for 24 hrs followed by a 12-hr exposure to5-FU 1000 μM. Cells were then processed for labeling by ApoptosisDetection Kit (Promega) according to the manufacturer's instructions.For quantitation of apoptosis, cells were plated at 250,000 cells/platein 6-well plates overnight, followed by 12-hr incubation with thefollowing chemotherapy agents: 5-FU 1000 μM, CPT-11 200 μM, cisplatin100p4, and etoposide 10 μM. Cells were collected by using a nonenzymaticcell dissociation medium (Sigma), washed with phosphate-buffered salineand subsequently stained for Annexin V and propidium iodide using anApoptosis detection kit (R & D Research) according to the manufacturer'sprotocol. The proportion of cells labeled with Annexin V and propidiumiodide was analyzed by XL Flow Cytometry Analyzer. Data was collectedfrom 100,000 events.

Transfection and Selection of Clone—The SPARC cDNA was cloned intopcDNA3.1 expression vector. Transfections were performed with 2.0 μg ofthe gene/expression vector construct using the polyethylenimine methodof Boussiff et al. (1995) with minor modifications (Tai et al., 2002).After transfection, cells were washed with phosphate-buffered saline(PBS, pH 7.4) and maintained in culture medium for 24 hours, followed bya change to an appropriate selection medium containing 1% Zeocin. Cellswere selected based on Zeocin resistance and individual colonies andclones from these colonies were then propagated for furtherverification. Stably transduced clones (MIP/SP) were screened for SPARCmRNA expression by reverse-transcription polymerase chain reaction(RT-PCR) analysis. MIP/SP clones with the highest expression of SPARCmRNA (by RT-PCR) and protein (by Western blot) were selected forsubsequent in-vitro and in-vivo studies. Control cell lines used forthis study included MIP101 cells stably transduced with pcDNA3.1 emptyvector only (MLP/Zeo) and selected based on Zeocin resistance.

Western Blot Analysis—Total protein was extracted from cell linescultured on 10-cm plates using CHAPS lysis buffer. 10-30 ug of totalprotein were electrophoresed using SDS-PAGE and transferred to PVDFmembrane. After blocking with 5% nonfat milk solution, the membraneswere incubated with anti-SPARC antibody (1:1000, HaematologicTechnologies Inc) overnight at 4° C. The membrane was subsequentlyincubated with rabbit anti-mouse HRP-conjugated secondary antibody(1:2000) for 1-hr at room temperature and detected by Extendi-Durachemiluminescence kit (Pierce). The same membrane was stripped usingWestern Blot Restore Stripping Buffer (Pierce) and subsequentlyre-probed for tubulin with a primary anti-tubulin mouse antibody (Sigma)and rabbit anti-mouse HRP-conjugated secondary antibody (1:2000) as aninternal control.

Immunohistochemistry—Paraffin sections of human colorectal cancers ornormal colonic epithelium were kindly provided by Dr. Maximo Loda (DanaFarber Cancer Institute, Boston). Prior to staining, the sections werewashed with 0.1% Tris-buffered saline (TBS) containing 0.1% Triton X-100(Sigma), treated with 1% H₂O₂ for 30 min, washed in TBS/0.1% Triton for30 min (×3) at room temperature, blocked with 3% BSA in TBS/0.1% Tritonfor 1 hr. Sections were then incubated with mouse anti-SPARC antibody(1:50) overnight at 4° C. (Haematologic Technologies Inc.), washedseveral times with TBS/Triton and counterstained withavidin-biotin-peroxidase (ABC) complex solution (Vecstain ABC kits,Vector Laboratories Inc, Burlingame, Calif.) for 1 hr, followed byincubation in DAB solution. Sections were mounted using Permount.

Colony forming Assay—For clonogenic cell survival studies, MIP101parental cells and MIP/SP cells were plated at 1,000 cells/plate in48-well plates and incubated with increasing concentrations of 5-FU (0,10 μM, 100 μM, 1000 μM), CPT-11 (0, 1 μM, 10 μM, 100 μM), or etoposide(0, 10 μM, 100 μM, 1000 μM) for 4 days. Cells were then washed with PBSand incubated in fresh medium containing the appropriate concentrationsof chemotherapy for an additional 7 days. Each well was stained withcrystal violet and the colonies with more than 50 cells were counted.The number of colonies formed in the treated group was calculated basedon the colonies formed from the control, untreated cells.

Concentrated SPARC-containing supernatant [SPARC(s)]—MIP/SP cells wereplated at 1×10⁶ cells in 100 cm flasks in DMEM (10% FBS, 1%penicillin/streptomycin, 0.1% Zeocin) for 24 hrs. Cells weresubsequently washed with PBS twice and incubated in serum-free VP-SFMmedium supplemented with glutamine 4 mM (Invitrogen) for 72 hrs. Thismedium was concentrated from 500 ml to 2 ml using Centricon Filter units(Millipore) at 4° C. All media collected and processed by this methodwere used for subsequent animal studies.

Animal Studies—Tumor xenograft animal models were used to assess theeffect of SPARC on tumor progression in-vivo. NIH nude mice (6 weeksold, Taconic Laboratories) were engrafted following subcutaneousinjection of 2×10⁶ cells into the left flank. Treatment regimens wereinitiated once the average tumor size was 50-75 mm³ in size. Tumormeasurements were performed using a hand-held caliper (Fisher) twiceweekly and weight measurements were made concurrently until thecompletion of the study. Chemotherapy was provided using a 3-week cycleregimen for a total of 6 cycles: 5-FU 25 mg/kg or CPT-11 25 mg/kgintraperitoneal injections three times on week 1 of each cycle, followedby 2 weeks of treatment-free periods. Dosing schedule for SPARC(s) was100 μL of SPARC(s) three times per week until the completion of thechemotherapy cycle.

Example 2 SPARC Expression in Chemotherapy Resistant Cells

Two chemotherapy resistant clones (MIP-5FUR and MIP-ETOR), as supportedby colony formation assays (FIG. 3) and TUNEL assay (FIG. 4), were usedfor the detection of SPARC in chemotherapy resistant cells. Microarrayanalysis identified a number of genes underexpressed in the resistantcells, including SPARC.

Underexpression at the gene expression level also translated into lowerlevels of SPARC protein levels in the chemotherapy resistant cell lines(FIG. 5A). This feature was not unique to the resistant cell linesdeveloped solely for the purposes of the current study, since anotherwell established uterine sarcoma cell line, MES-SA, also showeddecreased expression of SPARC when it is resistant to a differentchemotherapeutic agent, doxorubicin. (FIG. 5B). Furthermore, in normalhuman pathological samples, SPARC protein expression appears to behighest in the villi, with a decreasing gradient towards colonic crypts.This variable expression is lost in malignancy, with a general decreasein expression of SPARC in colorectal adenocarcinoma of various stages.

FIG. 19 shows human SPARC mRNA and protein levels in colorectal cancercell lines sensitive and resistant to chemotherapy. (A) Oligonucleotidemicroarray cluster analysis diagram (left panel) reveals that SPARC geneexpression is significantly lower in cell lines resistant tochemotherapy, which was confirmed by semi-quantitative RT-PCR (rightpanel). (B) Detection of SPARC expression levels in a paired uterinesarcoma cell line sensitive to chemotherapy (MES-SA) and resistant todoxorubicin (MES-SA/DX5) shows a similar decrease in expression in theresistant cell line. In breast cancer cell lines, MDA435 had slightlyhigher levels of SPARC expression than MCF-7. Low levels of expressionwere detected in pancreatic cancer cell line (CRL 1420), lung cancercell line (JMN 1B), colorectal cancer lines (RKO, CCL 227, HT 29). Highlevels of SPARC expression was detected in normal colon cell line (CRL1541) and a colon cancer cell line (HCT 116). (C) SPARC proteinexpression verifies that there is a significant decrease in this proteinin the MIP101 resistant clones (resistant cell lines: MIP/5FU, MIP/CPT,MIP/ETO, MIPT/CIS) in comparison to the normal parental cell line (lane5, MIP101). Similarly, another set of resistant cell line of uterinesarcoma origin (MES-SA/DX5, uterine sarcoma resistant to doxorubicin)shows decreased expression of SPARC in comparison to the parentalsensitive cell lines (MES-SA, parental uterine sarcoma).

FIG. 20 shows SPARC protein expression in human colonic epithelium. (A)Normal colon shows a differential pattern of SPARC protein expressionwith higher levels of the protein within the superficial cells proximalof the lumen and a gradient of decreasing expression towards the crypts.SPARC protein levels in (B) Adenocarcinoma of the colon, (C) mucinousadenocarcinoma and (D) adenocarcinoma of the colon metastatic to livershow low level of SPARC protein diffusely within the malignantepithelium. Sections 6 μm cross sections, ×20 magnification.

Example 3 SPARC Polypeptide Sensitizes Resistant Cells to 5-FU Treatment

In order to further delineate this potential role, we assessed theresponse of the resistant MIP101 cells (FIG. 6) to exogenous SPARC inreversing the resistant phenotype. As indicated by initial experiments,MIP101 cells resistant to 5-FU (MIP-5FUR) could not be triggered toundergo apoptosis with 5-FU at a concentration of 500 uM, while asignificant number of cells from the parental, sensitive cell lineunderwent apoptosis following exposure to a similar concentration of5-FU. A significant finding was observed with exogenous exposure ofresistant cells with SPARC: incubation of the resistant clones withSPARC for a 24-hr period followed by a 12-hr exposure to achemotherapeutic agent was sufficient in reversing the resistantphenotype, as apoptotic cells were once again detected by TUNEL assay incells exposed to concentrations of chemotherapy that previously did notstimulate cell death. Incubation with exogenous SPARC alone withoutsubsequent exposure to chemotherapy did not induce apoptosis in eitherthe parental MIP 101 or the resistant cells.

FIG. 21 shows assessment of the effect of SPARC in influencing thesensitivity of cells to chemotherapy. (A) Effect of exposure of MIP/5FUcells to exogenous SPARC in combination with 5-FU in-vitro. Assessmentof apoptosis by TUNEL assay shows positively stained cells in sensitiveMIP101 cells exposed to 5-FU 1000 uM (a, TUNEL stain; b, DAPI stain) butlack of apoptosis in the resistant phenotype (c, TUNEL stain; d, DAPIstain) following exposure to a similar concentration of 5-FU. However,following a 24 hr exposure to SPARC (5 ug/ml), 5-FU resistant cells onceagain became sensitive to 5-FU 1000 uM as shown by TUNEL-positivestained cells (e; f, DAPI stain), indicating the presence of apoptoticcells. This is the first indication that exogenous exposure to SPARCreverses the resistant phenotype of the 5-FU resistant cells and therebysuggesting that SPARC may function as a chemotherapy sensitizer. (B)Stably transduced MIP101 cell lines overexpressing SPARC (MIP/SP) andcontrol (MIP/Zeo) exposed to increasing concentrations of chemotherapy(5-FU, CPT-11 and etoposide) showed fewer cell colonies of MIP/SP cellswhen exposed to lower drug concentrations than MIP/Zeo cells, therebyindicating increased sensitivity of the SPARC overexpressing clones tochemotherapy as fewer cells survived at relatively lower concentrationsof chemotherapy. (B) Greater number of MIP101 overepressing SPARC(MIP/SP) undergo apoptosis following a 12-hr exposure to variouschemotherapeutic agents (ETO=etoposide, CIS=cisplatin,5-FU=5-fluorouracil, CPT=CPT-11) in comparison to control cells(MIP/Zeo) (p<0.05). Analysis of apoptosis following Annexin V labelingby flow cytometry represent results of three independent studiesperformed in triplicate. Results of the clonogenic assay (B) is arepresentative experiment that was repeated three times with similarresults.

Example 4 SPARC Polynucleotide Sensitizes Recombinant Cells to VariousChemotherapy Treatment

In order to test this hypothesis, MIP101 cells were transfected withSPARC for the purposes of generating overexpression systems foradditional in vitro studies. Two clones overexpressing SPARC (clones 4,5; FIG. 7) were used for subsequent studies.

The sensitivity of the SPARC-transfectants to various chemotherapeuticagents was assessed by colony formation assay, which showed that clonesoverexpressing SPARC were unable to form tumorigenic colonies at higherconcentrations of chemotherapy when compared to the parental cell lines.Similarly, FACS analysis of cell populations induced to undergoapoptosis following exposure to chemotherapeutic agents showed adramatic shift toward early apoptosis in SPARC-transfectants (FIG. 8D)following a 12-hr exposure to chemotherapy. A smaller population ofcells from the parental cell line underwent apoptosis followinginduction with chemotherapy only (FIG. 8C). Overall, there appeared tobe at least a 2-fold increase in the population of SPARC-overexpressingcells undergoing apoptosis in comparison to the parental MIP101 cellline following exposure to various chemotherapeutic agents (FIG. 9).FIG. 10 shows the response of SPARC transfectants to chemotherapyagents.

Example 5 SPARC Sensitizing is Observed In Vivo

The increased sensitivity to chemotherapy in vitro translated to thein-vivo model system, two of four animals showing complete tumorregression in animals transplanted with SPARC-transfectants following 6cycles of chemotherapy (FIG. 11). The remaining animals engrafted withSPARC-transfectants had a dramatic reduction in tumor growth rate incomparison to animals engrafted with the parental MIP101. All controlanimals (xenografts of MIP101 treated with chemotherapy) had tumors >400mm² by 50 days post initiation of chemotherapeutic treatment, whileanimals engrafted with SPARC-transfectant that did not undergo completetumor regression, had tumors that remained <300 mm² 140 dayspost-initiation of chemotherapy (result not shown).

Example 6 Method of Screening for an Agent which Modulates a SPARCPolypeptide Expression

The screening of a modulator of SPARC polypeptide expression can beperformed as a simple mammalian cell-based screen. A mammalian tissueculture cell line, e.g., Hela cells are first preincubated with randomcandidate small molecules. Cell clones are then screened usinganti-SPARC western blots or ELISA. Alternatively, a RT-PCR reaction iscarried out to examining the modulation on SPARC mRNA expression.

Example 7 Additional Animal Model Therapy

Various animal model therapy was carried out and the results are shownin FIG. 14-.

In FIG. 14, xenograft animals with tumors engrafted with either MIP101or MIP/SP treated with different chemotherapeutic agent (5-FU or CPT-11)show a more rapid rate of tumor regression of tumor xenografts of MIP/SPin comparison to tumor xenografts of the parental MIP101 cell line(MIP). Two of four animals carrying MIP/SP xenografts had complete tumorregression, while the remaining two had a much slower rate of tumorgrowth in comparison to the control animals carrying MIP101 exposed to asimilar treatment regimen. Representative animals with a tumorxenografts of MIP-SPARC treated with 2 cycles of 5-FU had completeremission by 23 days post-transplant or significantly smaller tumorsfollowing only 2 cycles of CPT-11 in comparison to an animaltransplanted with a xenograft of the parental MIP101.

In FIG. 15, more animals with xenografts of MIP/SP cells showed evidenceof complete tumor regression earlier in the post-radiation treatmentperiod than animals with xenografts of control MIP/Zeo cells. By 15weeks after radiation therapy, none of the MIP/SP xenograft animals hadevidence of tumor, while 30% of MIP/Zeo xenografts continued to harbortumors (n=10 animals/group; total dose of radiation: 100Gy).

In FIG. 16, combination treatment with SPARC(s) (IP, intraperitoneal)and 5-FU resulted in tumor regression that was significantly greaterthan treatment with 5-FU alone by 51 days after initiation of treatment.(B) This combination treatment of SPARC(s) (IP) and 5-FU resulted incomplete tumor regression in several animals by 84 days post-treatment,while this was not observed in animals treated with 5-FU alone.(mean±SE, n=6 animals/group).

In FIG. 17, combination treatment with SPARC(s) (SC, subcutaneous) and5-FU resulted in tumor regression that was significantly greater thantreatment with 5-FU alone throughout the treatment period. Thiscombination treatment of SPARC(s) (SC) and 5-FU resulted in completetumor regression in several animals by 42 days post-treatment, whilethis was not observed in animals treated with 5-FU alone. (mean±SE, n=6animals/group).

In FIG. 18, animals engrafted with MIP/5FU resistant cells were treatedwith either 5FU alone or combination SPARC(s) and 5-FU showed that rapidtumor growth continued in animals treated with 5-FU alone, whiledramatic tumor regression was observed in animals treated with thecombination therapy beginning at 28 days post-treatment. Several animalsreceiving combination SPARC(s) and 5FU therapy showed complete tumorregression by 117 days post-treatment. (mean±SE, n=6 animals/group).

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1.-23. (canceled)
 24. A composition for in vivo sensitizing a mammalcomprising an isolated SPARC family polynucleotide and a chemotherapyagent.
 25. The composition of claim 24, wherein the isolated SPARCfamily polynucleotide is carried in a virus or liposome.
 26. Thecomposition of claim 24, wherein the isolated SPARC familypolynucleotide and the chemotherapy agent are carried in a liposome. 27.The composition of claim 24, further comprising a pharmaceuticallyacceptable carrier.
 28. The composition of claim 24, wherein thechemotherapy agent is one or more of actinomycin D, adriamycin,altretamine, asparaginase, bleomycin, busulphan, capecitabine,carboplatin, carmustine, chlorambucil, cisplatin, CPT-11,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, doxorubicin,epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, fosfamide, irinotecan, liposomal doxorubicin,lomustine, melphalan, mercaptopurine, methotrexate, mitomycin,mitozantrone, oxaliplatin, procarbazine, steroids, streptozocin, taxol,taxotere, taxotere, tamozolomide, thioguanine, thiotepa, tomudex,topotecan, treosulfan, vinblastine, vincristine, vindesine orvinorelbine.
 29. The composition of claim 28, wherein the chemotherapyagent includes a taxol.
 30. The composition of claim 28, wherein thechemotherapy agent includes fluorouracil.
 31. The composition of claim24, wherein said isolated SPARC family polynucleotide encodes apolypeptide comprising an amino acid sequence selected from: SMOC-1,SPARC, hevin, SC1, QR-1, follistatin-like proteins (TSC-36) or testican.32. The composition of claim 31, wherein said isolated SPARC familypolynucleotide encodes a polypeptide comprising SEQ ID NO: 1 [the maturewild type SPARC protein].
 33. A method for in vivo sensitizing a mammaldiagnosed with cancer to a chemotherapy agent, said method comprising:administering therapeutically effective amounts of an isolated SPARCfamily polynucleotide and a chemotherapy agent.
 34. The method of claim33, wherein the isolated SPARC family polynucleotide is carried in avirus or liposome.
 35. The method of claim 33, wherein the isolatedSPARC family polynucleotide and the chemotherapy agent are carried in aliposome.
 36. The method of claim 33, further comprising administering apharmaceutically acceptable carrier.
 37. The method of claim 33, whereinthe chemotherapy agent is one or more of actinomycin D, adriamycin,altretamine, asparaginase, bleomycin, busulphan, capecitabine,carboplatin, carmustine, chlorambucil, cisplatin, CPT-11,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, doxorubicin,epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, fosfamide, irinotecan, liposomal doxorubicin,lomustine, melphalan, mercaptopurine, methotrexate, mitomycin,mitozantrone, oxaliplatin, procarbazine, steroids, streptozocin, taxol,taxotere, taxotere, tamozolomide, thioguanine, thiotepa, tomudex,topotecan, treosulfan, vinblastine, vincristine, vindesine orvinorelbine.
 38. The method of claim 37, wherein the chemotherapy agentincludes a taxol.
 39. The method of claim 37, wherein the chemotherapyagent includes fluorouracil.
 40. The method of claim 33, wherein saidisolated SPARC family polynucleotide encodes a polypeptide comprising anamino acid sequence selected from: SMOC-1, SPARC, hevin, SC1, QR-1,follistatin-like proteins (TSC-36) or testican.
 41. The method of claim40, wherein said isolated SPARC family polynucleotide encodes apolypeptide comprising SEQ ID NO: 1 [the mature wild type SPARCprotein].
 42. The method of claim 33, wherein said mammal exhibitsresistance to said chemotherapy agent.
 43. The method of claim 33,wherein the mammal is a human patient.